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    • 专利摘要:
    Method and application of preparing a medical food raw material for malignant tumors of probiotic fermented kelp: (1) preparing kelp powder; (2) perform the first fermentation; (3) inoculate with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, carry out the second fermentation; (4) separate the liquid and the solid of the second fermented drink, dry and pulverize the supernatant, get the dry supernatant powder, dry the precipitate at low temperature and spray, get the dry precipitate powder, mix the dry powder of the supernatant and precipitate, obtain the final product.
    公开号:BE1026585B1
    申请号:E20195597
    申请日:2019-09-10
    公开日:2020-10-28
    发明作者:Ting Wang；Ruiming Wang；Zhenshang Xu；Wu Meng；Yan Liang
    申请人:Univ Qilu Technology；
    IPC主号:
    专利说明:

    Description Method and application of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics
    Technical Field of the Invention The invention relates to a method and an application for preparing a medical food raw material for malignant tumors of fermented kelp using probiotics, in particular relates to a method of high activity , suitable for medical food raw materials for malignant tumors by extracting nutrient and anti-tumor active ingredients from fermented kelp using probiotics, it belongs to the technical field of extracting functional and plant foods.
    STATE OF THE PRIOR ART Algae are an important marine resource with a wide variety of species.
    In China, the seaweed is mainly kelp.
    Kelp, also known as Kombu and Lunbu, is often used as a food product and raw material for Chinese medicine, it is recorded in the Compendium of Materia Medica, Shennong Materia Medica, Edible Meteria Medica and the Compendium national Chinese herbal medicine.
    Kelp is not only rich in protein, vitamins and minerals, but also contains alginic acid, fucoidan, starch from brown algae, fibers from brown algae, mannitol, laminin, highly unsaturated fatty acid, fucoxanthin and sterol compounds rich in physiologically active substances.
    Among these, fucoidan is said to have immunomodulatory, anti-tumor, antiviral, antibacterial, anti-inflammatory, anti-oxidant, anticoagulant, and hypoglycemic effects according to reports, given the fact that a 4% content of fucoidan can prevent the growth of cancer cells and cause apoptosis of tumor cells, fucoidan has aroused great public interest.
    Studies have shown that fucoidan inhibits the growth of liver cancer cells QGY7703 in the logarithmic growth phase, thereby inhibiting the growth of tumors, the rate of inhibition of fucoidan on Hepes can exceed 50% without affecting the normal growth of the liver. mice, which showed superiority over the positive drugs.
    Algal polyphenols constitute a large category of compounds with different structures, and possess various biological activities, today there are many studies on the antitumor, antibacterial, antiviral and antioxidant activity of algal polyphenols. Among them, the Phlorotannins in Sargassum thunbergii have a strong anti-tumor effect on the cell line in human hepatoma BEL-7402 and on the cell line in human lung cancer A-549, the optimum inhibitory concentration is about
    0.085 to 0.10 mgeml / L; the raw extract of Leathesia nana has selective cytotoxic activity against KB cancer cells and HT-29 cancer cells and is basically non-toxic to normal NIH-3T3 cells. Yet at present, the development of kelp in China is mainly focused on extracting the feed from kelp, seaweed fertilizer and chemical raw materials such as iodine, alginic acid and mannitol, which are all part of the surface treatment of algae, with low technical content and low added value, the deep treatment of algae is still missing, and the active substances contained in the algae are not used in any way. efficient and comprehensive manner. Therefore, there is a need to continuously expand the scope of algae research and vigorously strengthen algae research in edible, medical, hygienic, forage, fertilizer and alginate industry, in order to have a more complete use of algae resources. Korean Patent Document KR 1020140057838A
    (No.KR1020120124100) publishes a fermentation product prepared by fermenting a mixture of lactic acid bacteria with other raw materials such as kelp and brown rice, said fermentation product has the function of promoting the decomposition of body fat, to promote defecation etc, and is useful in preventing diseases such as colon cancer and obesity.
    Food Formula for Special Medical Use (FSMP) is a special food category developed to provide nutritional support for certain diseases or people with special health problems, this has a positive effect on improving the therapeutic effect disease and postoperative recovery, the nutritional status of the patient, the resistance of his own body as well as the overall health of the patients.
    In China, the FSMP industry is in its infancy, with less focused research and a lower degree of industrialization, a market long occupied by foreign food products, it finds itself in a situation of low quantity and price Student.
    In order to improve the scientific and technological innovation capacity of special medical use preparations in China and promote the conversion of new and old kinetic energies of medical care and health industry, there is an urgent need for research on medical food process technology, in particular on the development of medical food raw material preparation technology.
    Disclosure of the Invention In view of the foregoing, an object of the present invention is to provide a method and application for the preparation of a medical food raw material for malignant tumors of fermented kelp using probiotics. The food raw materials extracted by the process have high safety, targeted effect and produce no pollution, their contents of nutrients and active and nutritional substances are high, and have good tumor inhibiting effect and high stability.
    The technical solution of the present invention is as follows: A method of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics, comprises the following steps: (1) spraying the kelp to produce the kelp powder; (2) Add the kelp powder obtained in step (1) to the primary fermentation medium, mix evenly, then insert the solution of Bacillus bacteria, ferment once to remove Bacillus cells, thus preparing the first fermented drink of the kelp;
    Said Bacillus is one or a combination of two or more Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis and Bacillus coagulans; (3) inoculating the first fermented kelp drink obtained in step (2) with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, performing the second fermentation to obtain the second fermented kelp drink; Said lactic acid bacteria is one or a combination of two or more of Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus gasseri, Lactobacillus acidophilus and Lactobacillus crispatusv; (4) The second fermented drink obtained in step (3) is subjected to solid-liquid separation, and the supernatant is spray dried to obtain the dry powder of the supernatant, the precipitate is dried at low temperature, and then pulverized. To obtain the dry precipitate powder, mix the dry supernatant powder and the dry precipitate powder to obtain the final product.
    According to a preferred embodiment of the present invention, in step (1), said kelp is fresh kelp or kelp expanded by water, after desalting it is dried until having a lower water content or equal to 5%.
    According to another preferred embodiment of the present invention, the desalting step is to rinse 2 to 5 times with pure water.
    According to another preferred embodiment of the present invention, the water expansion process is as follows: the kelp is expanded with 10 to 20 times of pure water and at a temperature of 20 to 40 ° C for 2 at 7 O'clock.
    According to another preferred embodiment of the present invention, the drying is carried out by a drying process and the drying temperature is 45 to 90 ° C.
    According to a preferred embodiment of the present invention, in step (1), the diameter of the sprayed particles is between 10 to 1000 µm.
    According to a preferred embodiment of the present invention, step (1) further comprises a sterilization step after spraying, advantageously sterilization is carried out by irradiating 1000 to 2500 rads for 1.5 to 5 hours ; more preferably, the irradiation sterilization time is 3 hours.
    According to a preferred embodiment of the present invention, in step (2), the condition of the primary fermentation is as follows: ferment at a temperature of 35 to 38 ° C and at a rotational speed of 180 to 220 rpm for 24 to 48 hours.
    According to a preferred embodiment of the present invention, in step (2), the components of the primary fermentation medium are as follows, all in percentage by weight: 1.5 to 2.5% glucose, 1.0 to 2.0% peptone, 0.04 to 0.06% chloride, 0.04 to 0.06% beef paste and the remainder in water.
    According to a preferred embodiment of the present invention, in step (2), the amount of kelp powder added is 7 to 12% of the mass of the primary fermentation medium.
    According to a preferred embodiment of the present invention, in step (2), the amount of insertion of the solution of Bacillus bacteria is between 1% and 5% of the mass of the primary fermentation medium, and the concentration bacteria of the solution of Bacillus bacteria is between 1.5 to 7.5x10 / ° cfu / mL.
    According to a preferred embodiment of the present invention, in step (2), the Bacillus spores are removed by centrifugation and are centrifuged at 7000 to 10,000 rpm for 8 to 12 minutes.
    The aforementioned seaweed powder is degraded by various enzymes produced by the growth of Bacillus, and the viscosity of the fermented drink gradually increases, so that a solid-liquid separation is necessary not to affect the subsequent fermentation. According to a preferred embodiment of the present invention, in step (3), the amount of insertion of the lactic acid bacteria is between 4 to 6% of the volume of the first fermented drink of the kelp, and the bacterial concentration of the bacteria lactic acid is between 2.5 to 9.5x10 / ° cfu / mL.
    According to a preferred embodiment of the present invention, in step (3), rest the second fermentation at a temperature of 28 to 33 ° C for 36 to 72 hours.
    According to a preferred embodiment of the present invention, in step (4), the solid-liquid separation is carried out at a speed of 7000 to 10,000 rpm for 10 to 20 minutes.
    According to a preferred embodiment of the present invention, in step (4), the spray drying conditions are as follows: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C , the spray air pressure is 0.1 Mpa, and the dry air volume is 0.5 m / L; low temperature drying temperature is 45-65 ° C.
    According to a preferred embodiment of the present invention, in step (4), before the solid-liquid separation step, it further comprises the post-maturation step: placing the second fermented beverage of the kelp in a state sealed at 8 to 12 ° C, then mature for 15 to 30 days to obtain the final product.
    Post-maturation has the effect of stabilizing the active components in the fermented beverage by post-maturation at low temperature.
    According to a preferred embodiment of the present invention, in step (4), said spray is the ultrafine spray.
    The above is the application of a medical food raw material for malignant tumors in the preparation of health foods or drugs for malignant tumors.
    Beneficial effect
    1. The present invention prepares for the first time a medical food raw material for malignant tumors by the second fermentation of kelp with the help of probiotics, to release nutrients and active components contained in kelp, its method of preparation is safe, reliable, environmentally friendly and efficient, while avoiding the use of chemical reagents such as strong acid and strong alkali to reduce polluting emissions, the fermented drink contains dietary fiber in order to achieve a discharge without slag in accordance with the policy of green environmental protection and conversion of new and old kinetic energies.
    2. The medical food raw material for malignant tumors prepared by the method of the invention contains a high content of fucoidan, polyphenol and fucose which is closely related to the antitumor, antiviral, antibacterial, anti-inflammatory and antioxidant effects, and the drink fermented kelp prepared in the embodiment shows a good inhibitory effect on the growth of glioma cells; at the same time, the present invention fully preserves and releases the active components in kelp, said fermented probiotics can produce active ingredients such as glutathione, gamma aminobutyric acid, extracellular cellulase, pectinase etc, increase the type and content of active ingredients in the fermented product, and improve the nutritional and curative effect of raw materials of food preparations for tumors.
    Brief Descriptions of the Drawings Fig. 1 is a histogram for comparing polyphenol extraction rates of the first embodiment with the first comparative embodiment and the second comparative embodiment; Fig. 2 is a standard graph showing the results of the detection of the polyphenol content in the first fermented kelp drink of the first embodiment;
    Fig. 3 is an electron micrograph showing the inhibitory effects of the medical food raw material for malignant tumors on U87 / LC3 glioma cells in the second embodiment;
    Among them: a. the electron microscopic structure of U87 / LC3 cells at 0 hours after use; b. the electron microscopic structure of U87 / LC3 cells at 4 hours after use; vs. the electron microscopic structure of U87 / LC3 cells 8 hours after use; d. electron microscopic structure of cells
    U87 / LC3 at 12 hours after use; e. the electron microscopic structure of U87 / LC3 cells at 16 hours after use; Descriptions of Preferred Embodiments Technical solutions of the present invention are described in more detail below in conjunction with the drawings and embodiments of the present invention, but the scope of protection of the present invention is not. not limited to that.
    All the technical and scientific terms used here have the same meaning as that commonly understood by those skilled in the art to which the present invention relates, unless otherwise indicated.
    Source of Biomaterials The source of lactic acid bacteria in the present invention is not limited, and lactic acid bacteria from the food source of the present invention can be used; Lactobacillus plantarum whose deposit number is CICC 20261, Lactobacillus casei whose deposit number is CICC 20277, Lactobacillus acidophilus whose deposit number is CICC 22150 and Streptococcus thermophilus whose deposit number is CICC 20370 are all purchased from China Center of Industrial Culture Collection, CICC; Lactobacillus gasseri with deposit number JCM 11657 and Lactobacillus crispatusv with deposit number JCM 2009 are all purchased from the Japan collection of microorganisms.
    The Bacilli in the present invention are all probiotics, and the source of the Bacilli is not limited in the present invention, the Bacilli derived from the food source of the present invention can be used; Bacillus subtilis involved in embodiments and comparative embodiments purchased from China Center of Industrial Culture Collection, CICC, whose accession number CICC 24434; Bacillus pumilus whose deposit number is CGMCC
    1.7925, Bacillus licheniformis whose deposit number is CGMCC
    1.5832, Bacillus coagulans whose deposit number is CGMCC
    1.10832 are all purchased from the China General Microbiological Culture Collection Center, CGMCC; The experimental materials used in the embodiments are all materials sold in conventional commerce and can be purchased through commercial channels. The spraying step is carried out using the micro-sprayer of Jiangsu Mingfeng Machinery Manufacturing Co, Ltd., an ordinary commercial product; The spray drying equipment is the EU brand OM-1500 / OM-1500A laboratory spray dryer, the spray drying conditions are defined as: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C, the spray air pressure is 0.1 Mpa, and the dry air volume is
    0.5m "/ L; an ordinary commercial product; The drying stage adopts the GZX-9240MBE digital display blow-molding oven from Shanghai Boxun Industrial Co., Ltd. medical equipment factory, the low-temperature drying temperature is 45-65 ° C, and the water content is less than 3%; an ordinary commercial product; Ultrafine spray adopts Beijing Kaichuang Tonghe Technology Development Co., Ltd. KC-701 ultrafine vegetable sprayer, an ordinary commercial product; The mixing stage adopts the V-type mixer produced by Changzhou Qianjiang Drying Equipment Factory, an ordinary commercial product.
    Detection method Fucoidan: detected according to the method described in Lui Haiguang, 2004, Separation, purification and activity of laminaria polysaccharide [D]. Shandong University; Fucose: detected according to the method of GB / 5009.168-2016, Agricultural Industry Standard of the People's Republic of China
    NY / T2279-2202; Phlorotannins: detected according to the method described in Yang Huicheng, 2008, the extraction and separation of polyphenols from Laminaria japonica Aresch and research on their antitumor and antibacterial activities, [D]. China Ocean University; Polysaccharides: detected according to the phenol and sulfuric acid method described in SNT 4260-2015 Determination of crude polysaccharides in exported foods of plant origin; Tumor cell inhibition assay: based on the method described in Yang Huicheng, 2008, the extraction and separation of polyphenols from Laminaria japonica Aresch and research on their antitumor and antibacterial activities, [D]. China Ocean University; First Embodiment A method of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics comprises the following steps: (1) spraying the kelp having a water content of 5% or less up to a particle size of 500um, and sterilize to obtain the kelp powder; said kelp is fresh kelp, and after being washed 3 times with pure water, it is dried at a temperature of 70 ° C.
    The sterilization step is as follows: sterilize at 1500 krad for 3 hours;
    (2) add the kelp powder obtained in step (1) to the primary fermentation medium, the amount of added kelp powder is 10% of the mass of the primary fermentation medium, mix the kelp powder evenly, then insert the solution of Bacillus bacteria, carry out the first fermentation for 36 hours at a temperature of 36 ° C and at a rotational speed of 200 rpm, the Bacillus cells are removed by centrifugation at 8500 rpm for 10 minutes to prepare the first fermented drink from kelp;
    Said Bacillus is Bacillus Pumilus; Said components of the primary fermentation medium are as follows,
    all in percentage by weight:
    1.5% glucose, 1.0% peptone, 0.04% chloride, 0.04% beef paste and the rest in water;
    Said insertion amount of the solution of Bacillus bacteria is 3% of the mass of the primary fermentation medium, and the bacterial concentration of the solution of Bacillus bacteria is 5 × 10 ° cfu / mL;
    (3) inoculate the first fermented kelp drink obtained in step (2) with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, the amount of lactic acid bacteria is 5% of the volume of the first fermented kelp drink , the bacterial concentration of lactic acid bacteria is 3.0x10 "" cfu / mL, stand the fermented drink for 36 hours at a temperature of 30 ° C to obtain the second fermented drink of kelp; Said lactic acid bacteria are Lactobacillus plantarum; (4) The second fermented drink obtained in step (3) is centrifuged at 8500 rpm for 15 minutes, the supernatant is spray dried to obtain the dry powder of the supernatant, and the precipitate is dried at low temperature. temperature and sprayed ultra-fine in order to obtain the dry precipitate powder; mix the dry supernatant powder and the dry precipitate powder to obtain the final product.
    The spray drying conditions are as follows: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C, the spray air pressure is 0.1 Mpa, and the volume of dry air is 0.5m '/ L; the low temperature drying temperature is 55 ° C.
    The first fermented kelp drink prepared in step (2) was detected according to the method described in Fan Wenle, 2006, Global Kelp Use Research [D]. Tianjin University of Science and Technology, the results are shown in Table 1: Table 1 Content and composition of fucoidan in the first fermented drink of kelp Fucoidan | The The acid The Les (the rate | sulfate (g) | uronic (g) | viscosity polyphenols of extraction%) (the rate (kpa.s) | of extraction%) id 550
    Second Embodiment A method of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics comprises the following steps: (1) spraying the kelp having a water content of 5% or less to a particle size of 10um, and sterilize to obtain the kelp powder; said kelp is water-expanded kelp, and after being washed 5 times with pure water, it is dried at a temperature of 45 ° C; Said process of water-expanded kelp is as follows: put the dry kelp in 20 times of pure water and immerse at a temperature of 20 ° C for 7 hours; The sterilization step is as follows: sterilize at 1000 krad for 5 hours;
    (2) add the kelp powder obtained in step (1) to the primary fermentation medium, the amount of added kelp powder is 7% of the mass of the primary fermentation medium, mix the kelp powder evenly, then insert the solution of Bacillus bacteria, carry out the first fermentation for 48 hours at a temperature of 38 ° C and at a rotational speed of 180 rpm, the Bacillus cells are removed by centrifugation at 7000 rpm for 12 minutes to prepare the first fermented drink from kelp;
    Said Bacillus is a combination of Bacillus Pumilus and Bacillus coagulans;
    Said components of the primary fermentation medium are as follows, all in percent by weight:
    2.5% glucose, 2.0% peptone, 0.06% chloride, 0.04% beef paste and the rest in water; Said insertion amount of the solution of Bacillus bacteria is 1% of the mass of the primary fermentation medium, and the bacterial concentration of the solution of Bacillus bacteria is 7.5x10 "° cfu / mL; (3) inoculate the first fermented kelp drink obtained in step (2) with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, the amount of lactic acid bacteria is 4% of the volume of the first fermented kelp drink, the bacterial concentration of bacteria lactic acid is 45x10 "cfu / mL, stand the fermented drink for 48 hours at a temperature of 33 ° C to obtain the second fermented kelp drink; Said lactic acid bacteria is a combination of Lactobacillus plantarum and Lactobacillus crispatusv; (4) The second fermented drink obtained in step (3) is post-matured and centrifuged at 10,000 rpm for 10 minutes, the supernatant is spray-dried to obtain the dry powder of the supernatant, and the precipitate is dried at low temperature and sprayed ultra-fine to obtain the dry precipitate powder, mix the dry supernatant powder and the dry precipitate powder to obtain the final product.
    The spray drying conditions are as follows: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C, the spray air pressure is 0.1 Mpa, and the volume of dry air is 0.5m '/ L; the low temperature drying temperature is 65 ° C.
    The post-maturation step is as follows: Place the second fermented kelp drink in a sealed state at 8 ° C, and perform post-maturation for 30 days to obtain the final product.
    Post-maturation has the effect of stabilizing the active components in the fermented beverage by maturation at low temperature.
    Third Embodiment A method of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics comprises the following steps: (1) spraying the kelp having a water content of 5% or less Up to a particle size of 1000um, and sterilize in order to obtain the kelp powder; said kelp is kelp expanded by water, and after having been washed twice with pure water, it is dried at a temperature of 90 ° C; Said process of water-expanded kelp is as follows: put the dry kelp in 10 times of pure water and immerse at a temperature of 40 ° C for 2 hours; The sterilization step is as follows: sterilize at 2500 krad for 1.5 hours; (2) add the kelp powder obtained in step (1) to the primary fermentation medium, the amount of added kelp powder is 12% of the mass of the primary fermentation medium, mix the kelp powder evenly, then insert the solution of Bacillus bacteria, carry out the first fermentation for 24 hours at a temperature of 35 ° C and at a rotational speed of 220 rpm, the Bacillus cells are removed by centrifugation at 10,000 rpm for 8 minutes to prepare the first fermented drink from kelp; Said Bacillus is a combination of Bacillus subtilis, Bacillus Pumilus, Bacillus licheniformis and Bacillus coagulans; Said components of the primary fermentation medium are as follows, all in weight percent: 2.0% glucose, 1.5% peptone, 0.046% chloride, 0.05% beef paste and the rest water; Said insertion amount of the solution of Bacillus bacteria is 5% of the mass of the primary fermentation medium, and the bacterial concentration of the solution of Bacillus bacteria is 1.5x10 "° cfu / mL. (3) inoculate the first fermented kelp drink obtained in step (2) with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, the amount of lactic acid bacteria is 6% of the volume of the first fermented kelp drink, the bacterial concentration of bacteria lactic acid is 5.0x10 "cfu / mL, stand the fermented drink for 52 hours at 28-33 ° C to obtain the second fermented kelp drink; Said lactic acid bacteria is a combination of Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus gasseri, Lactobacillus acidophilus and Lactobacillus crispatusv; (4) The second fermented drink obtained in step (3) is post-matured and centrifuged at 10,000 rpm for 10 minutes, the supernatant is spray-dried to obtain the dry powder of the supernatant, and the precipitate is dried at low temperature and sprayed ultra-fine to obtain the dry precipitate powder, mix the dry supernatant powder and the dry precipitate powder to obtain the final product. The spray drying conditions are as follows: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C, the spray air pressure is 0.1 Mpa, and the volume of dry air is 0.5m '/ L; the low temperature drying temperature is 65 ° C.
    The post-maturation stage is as follows:
    Place the second fermented kelp drink in a sealed state of 8 ° C, and post-ripen for 30 days to obtain the final product. Post-maturation has the effect of stabilizing the active components in the fermented beverage by maturation at low temperature.
    First Comparative Embodiment Fucoidan is extracted by water extraction, acid extraction and enzymatic extraction according to the method described in Fan Wenle, 2006, Research on the global use of kelp [D ]. Tianjin University of Science and Technology. Second Comparative Embodiment The method described in the second embodiment differs in that the primary fermentation uses lactic acid bacteria and the second fermentation uses Bacillus. First experimental example The content of heteropolysaccharide and of fucoidan of different products prepared in the first embodiment and the first comparative embodiment is detected according to the method described in Fan Wenle, 2006, Research on the global use of kelp [D] . Tianjin University of Science and Technology, the results of which are shown in Table 1: Table 2 The comparison of the heteropolysaccharide and fucoidan content of the first embodiment and the first comparative embodiment The group the content of | the content of heteropolysaccharide fucoidan | (g) (8) Water extraction | 12.6 1.52 (90 ° C) Extraction
    13.4 2.05 enzymatic Acid extraction 1.38 The first mode of a 20.9 2.67 Carrying out As shown in Table 2, the content of heteropolysaccharide and fucoidan of the first embodiment is significantly increased in comparison with that of the 'hot water, acid and enzymatic extraction of the first comparative embodiment, the fucoidan content which would have immunomodulatory effects,
    antitumors, antivirals, antibacterials, anti-inflammatories, anti-oxidants, anticoagulants, and hypoglycemic agents is 1.93 times more than that in acid extraction, and 1.75 times more than that obtained in water extraction hot; The algae polyphenol content of different products prepared in the first embodiment and the first comparative embodiment is detected according to the method described in Fan Wenle, 2006, Research on the global use of kelp [D]. Tianjin University of Science and Technology, the results of which are shown in Figure 1; As shown in Fig. 1, the content of polyphenols of algae having clear anti-tumor activity in the second embodiment is 2.36 times that in the hot water extraction and 2.18 times that in the hot water extraction. the acid extraction of the first comparative embodiment, and 1.65 times more than that in the second comparative embodiment. The above data shows that the enzymatic hydrolysis sequences of different probiotics also have a significant effect on the polyphenol content of algae.
    Second experimental example The LC3 protein is a protein associated with microtubules which undergoes a specific change and a punctual structure of autophagy around it. The LC3 protein of the U87 / LC3 glioma cell line is labeled with green GFP fluorescence and can be observed under the fluorescence microscope.
    The effect of the product of the third embodiment on the U87 / LC3 glioma cell line is examined by the following procedure, as follows: (1) U87 / LC3 cells are plated in a small 5 cm glass petri dish. in diameter for an adherent culture, the cellular liquid in each dish is kept at 2 ml, and the incubator is cultured for 18 hours. (2) Add 2.5% (mass ratio) of the fermented drink of the third embodiment, respectively form 5 groups in parallel, including a blank control without adding the fermented drink of the third embodiment, after being put into culture for 4 hours, 8 hours, 12 hours and 16 hours, observe the changes by the fluorescence microscope. As can be seen in Fig. 3, when the U87 / LC3 glioma cells are added to the product obtained in the third embodiment, it is clearly observed by the fluorescence microscope that a point structure appears around the cells. Specifically, the GFP-LC3 protein changed significantly after adding the product obtained in the third embodiment, the point structure around U87 / LC3 after 4 hours of action is as shown in Figure 3b and in the Following periods of 8 hours to 12 hours, this point structure still exists and gradually increases, after 16 hours, the cell structure breaks down and the tumor cells are dead.
    This modification process clearly demonstrates that the product prepared in the third embodiment induces autophagy in the cells, i.e. the fermented kelp drink prepared in the third embodiment shows a good inhibitory effect on growth. glioma cells.
    权利要求:
    Claims (10)
    [1]
    A method of preparing a medical food raw material for malignant tumors of fermented kelp using probiotics, characterized in that it comprises the following steps: (1) pulverizing the kelp to produce the kelp powder; (2) Add the kelp powder obtained in step (1) to the primary fermentation medium, mix evenly, then insert the solution of Bacillus bacteria, ferment once to remove Bacillus cells, thus preparing the first fermented drink of the kelp; Said Bacillus is one or a combination of two or more Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis and Bacillus coagulans; (3) inoculating the first fermented kelp drink obtained in step (2) with lactic acid bacteria or a mixture of lactic acid bacteria and bifidobacterium, performing the second fermentation to obtain the second fermented kelp drink; Said lactic acid bacteria is one or a combination of two or more of Lactobacillus plantarum, Lactobacillus casei, Streptococcus thermophilus, Lactobacillus gasseri, Lactobacillus acidophilus and Lactobacillus crispatusv; (4) The second fermented drink obtained in step (3) is subjected to solid-liquid separation, and the supernatant is spray dried to obtain the dry powder of the supernatant, the precipitate is dried at low temperature, and then pulverized. To obtain the dry precipitate powder, mix the dry supernatant powder and the dry precipitate powder to obtain the final product.
    [2]
    2. Method according to claim 1, characterized in that, in step (1), the kelp is fresh kelp or kelp expanded by water, after desalting it is dried until having a content of water less than or equal to 5%; more preferably, the desalting step is to rinse 2 to 5 times with pure water; more preferably, said water expansion method is as follows: the kelp is expanded by 10 to 20 times of pure water and at a temperature of 20 to 40 ° C for 2 to 7 hours; more preferably, said drying is carried out by a drying process and the drying temperature is 45 to 90 ° C.
    [3]
    3. Method according to claim 3, characterized in that the diameter of the particles sprayed in step (1) is between 10 to 1000 µm; preferably step (1) further comprises a sterilization step after spraying, more preferably sterilization is carried out by irradiating 1000 to 2500 rads for 1.5 to 5 hours; more preferably, the irradiation sterilization time is 3 hours.
    [4]
    4. Method according to claim 1, characterized in that, in step (2), the condition of the primary fermentation is as follows: ferment at a temperature of 35 to 38 ° C and at a speed of rotation of 180 to 220 rpm for 24 to 48 hours; preferably, the components of the primary fermentation medium are as follows, all in weight percent: 1.5 to 2.5% glucose, 1.0 to 2.0% peptone, 0.04 to 0.06 % chloride, 0.04 to 0.06% beef paste and the rest in water.
    [5]
    5. Method according to claim 1, characterized in that, in step (2), the amount of kelp powder added is 7 to 12% of the mass of the primary fermentation medium; preferably, in step (2), the amount of insertion of the solution of Bacillus bacteria is between 1% and 5% of the mass of the primary fermentation medium, and the bacterial concentration of the solution of Bacillus bacteria is between 1.5 to 7.5x 10 "° cfu / mL.
    [6]
    6. Method according to claim 1, characterized in that, in step (2), the Bacillus spores are removed by centrifugation and are centrifuged at a speed of 7000 to 10,000 rpm for 8 to 12 minutes.
    [7]
    7. Method according to claim 1, characterized in that, in step (3), the amount of insertion of lactic acid bacteria is between 4 to 6% of the volume of the first fermented drink of the kelp, and the bacterial concentration. lactic acid bacteria is between 2.5 to 9.5x10 "cfu / mL.
    preferably, in step (3), the second fermentation is carried out at a temperature of 28 to 33 ° C and is left for 36 to 72 hours.
    [8]
    8. Method according to claim 1, characterized in that, in step (4), the solid-liquid separation is carried out at a speed of 7000 to 10,000 rpm for 10 to 20 minutes; preferably, in step (4), the spray drying conditions are as follows: the inlet temperature is 90 ° C, the outlet temperature is 65 ° C, the air pressure of spraying is 0.1 Mpa and the dry air volume is 0.5m '/ L; low temperature drying temperature is 45-65 ° C.
    [9]
    9. The method of claim 1, characterized in that, in step (4), before the solid-liquid separation step, it further comprises the post-maturation step: Place the second fermented beverage of the kelp in a sealed state between 8 and 12 ° C, then mature for 15 to 30 days in order to obtain the final product. Post-maturation has the effect of stabilizing the active components in the fermented drink by post-maturation at low temperature; preferably, in step (4), said spray is the ultrafine spray.
    [10]
    10. The application of a medical food raw material for malignant tumors according to the method of claim 1 in the preparation of health foods or medicaments for malignant tumors.
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    法律状态:
    2020-12-04| FG| Patent granted|Effective date: 20201028 |
    优先权:
    申请号 | 申请日 | 专利标题
    CN201811103176.4A|CN109288042A|2018-09-20|2018-09-20|A kind of method and application using probiotics fermention kelp preparation malignant tumour medical food raw material|
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