CN109288042A - A method and application of using probiotics to ferment kelp to prepare medical food raw materials for malignant tumors - Google Patents
A method and application of using probiotics to ferment kelp to prepare medical food raw materials for malignant tumors Download PDFInfo
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Abstract
The present invention relates to a kind of methods and application using probiotics fermention kelp preparation malignant tumour medical food raw material.This method comprises: (1) crushes kelp, Kelp Powder is made；(2) one time fermentation culture medium is added in Kelp Powder, through one time fermentation, kelp first time fermentation liquid is made；(3) second of fermentation liquid of kelp is made in the Mixed Microbes of inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium into kelp first time fermentation liquid, secondary fermentation；(4) by second of fermentation liquid through being separated by solid-liquid separation, the spray-dried obtained supernatant dry powder of supernatant, precipitate through low temperature drying, after crushing, be made precipitating dry powder, mixing supernatant dry powder and precipitating dry powder to get.The mode that first passage of the present invention carries out prebiotic microzyme secondary fermentation to kelp prepares malignant tumour medical food raw material, and this method can discharge nutriment and active constituent in kelp.
Description
Technical field
The present invention relates to it is a kind of using probiotics fermention kelp preparation malignant tumour medical food raw material method and application,
In particular to a kind of high activity, suitable for malignant tumour medical food raw material using probiotics fermention extract kelp nutrition and
The method of anti-tumor active ingredient belongs to functional food and technical field of plant extraction.
Background technique
Seaweed species are various, resourceful, are important marine resources.In China, seaweed is mainly based on kelp.Kelp
Also known as thallus laminariae, synthetic fibre cloth, frequently as food and Chinese medicine material, Compendium of Material Medica, " legendary god of farming's book on Chinese herbal medicine position ", " edible book on Chinese herbal medicine " and " in
State's Chinese herbal medicine compilation " in it is on the books.Kelp not only proteins,vitamins,and minerals rich in, also containing such as brown alga
Acid, fucoidin, laminaran, brown alga fiber, mannitol, laminine, high unsaturated lipid boat acid, fucoxanthine and sterols
The physiological activator abundant such as compound.Wherein, fucoidin is reported with immunoregulation effect, antitumor action, resists
Virus function, antibacterial and anti-inflammation functions, antioxidation, the effect of anticoagulant rouge, hypoglycemic effect etc., wherein because fucoidin only accounts for
4% content prevents from growth of cancer cells, and causes apoptosis of tumor cells and cause people's extensive concern.Studies have shown that brown
Algae carbohydrate gum inhibits liver cancer cells QGY7703 to enter logarithmic growth phase, to contain the growth of tumour, fucoidin pair
The tumour inhibiting rate of Hepes can be more than 50%, and not influence the normal growth of mouse, to show the superiority than positive drug.
Seeweed polyphenol is the different compound of a big class formation, has multiple biological activities, at present to seeweed polyphenol anti-tumor activity, anti-
Bacterium antiviral activity, antioxidant activity research are more.Wherein, in sargassum thunbergii brown algae polyphenols to Human Hepatic Carcinoma Cell Line BEL-7402 and
Human lung cancer cell lines A-549 has stronger antitumor action, and optimal inhibition concentration is in 0.085~0.10mgm L/L or so；
Small mucous membrane algae crude extract has selecting cell cytotoxic activity to KB cancer cell, HT-29 cancer cell, to normal cell NIH-3T3 base
Do not have toxicity in sheet.
However, the exploitation of China kelp at present focuses primarily upon kelp food, seaweed (band) fertilizer and iodine, alginic acid, sweet
The extraction for revealing the industrial chemicals such as alcohol, belongs to the shallow processing of seaweed, with low content of technology, added value of product is low, still not to seaweed
Carry out deep processing, the active material in high-efficiency comprehensive utilization seaweed.The research range to seaweed thus must be constantly expanded, greatly
Power reinforces research of seaweed in terms of edible, medicinal, health care, feed, fertilizer and phycocolloid, so as to more sufficiently comprehensively
Utilize marine algae resource.
Korean patent document KR1020140057838A (application number KR1020120124100) discloses a kind of using kelp
The tunning prepared with other raw materials such as brown rice using mixing lactic acid bacteria fermentation, which, which has, promotes body fat point
Solution promotes the functions such as defecation, helps to prevent the diseases such as colorectal cancer and obesity.
Special medicine purposes formula food (FSMP) is to provide nutrition branch for certain diseases or special health status crowd
The Special food classification held and grown up, in the therapeutic effect and postoperative rehabilitation effect that improve disease, the nutrition for improving patient
Situation, enhancing body itself resistance, the horizontal aspect of holistic health for improving patient have good result.In China, FSMP is produced
Industry is in the starting stage, targetedly studies less, and industrialization level is lower, and market is occupied for a long time by foreign food, in amount
The high situation of few valence.To improve China's special medicine purposes formula food sci-tech innovation ability, doctor is pushed to support health industry new and old
Kinetic energy conversion needs the technology research for carrying out medical food, especially research and development medical food material prepared technology.
Summary of the invention
In view of this, preparing the medical food of malignant tumour using probiotics fermention kelp the purpose of the present invention is to provide a kind of
The method and application of product raw material.The raw-food material that this method is extracted is highly-safe, with strong points, pollution-free, while nutritional ingredient,
Activated species and content are high, and tumor inhibitory effect is good, and stability is strong.
Technical solution of the present invention is as follows:
A method of malignant tumour medical food raw material is prepared using probiotics fermention kelp, comprising the following steps:
(1) kelp is crushed, Kelp Powder is made；
(2) one time fermentation culture medium is added in Kelp Powder made from step (1), is uniformly mixed, then accesses bacillus
After bacterium solution, one time fermentation removes bacillus thallus, and kelp first time fermentation liquid is made；
The bacillus be bacillus subtilis, bacillus pumilus, bacillus licheniformis, in bacillus coagulans
A combination of one or more；
(3) inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium into kelp first time fermentation liquid made from step (2)
Second of fermentation liquid of kelp is made in Mixed Microbes, secondary fermentation；
The lactic acid bacteria be lactobacillus plantarum, Lactobacillus casei, streptococcus thermophilus, lactobacillus gasseri, lactobacillus acidophilus and
Curl the combination of one or more of lactobacillus；
(4) by second of fermentation liquid made from step (3) through being separated by solid-liquid separation, the spray-dried obtained supernatant dry powder of supernatant,
Precipitating through low temperature drying, after crushing, be made precipitating dry powder, mixing supernatant dry powder and precipitating dry powder to get.
Preferred according to the present invention, in the step (1), kelp is fresh kelp or the kelp after being soaked, after desalination,
It dries to water content≤5%.
According to the present invention it is further preferred that the desalination is to be rinsed 2~5 times using pure water.
According to the present invention it is further preferred that the process that is soaked is as follows: dry kelp is immersed the pure of 10~20 times of quality
In water purification, be soaked 2~7h under conditions of 20~40 DEG C.
According to the present invention it is further preferred that it is described it is dry use drying mode, 45~90 DEG C of drying temperature.
Preferred according to the present invention, in the step (1), powder particle diameter is 10~1000 μm.
It is preferred according to the present invention, in the step (1), further include the steps that sterilizing after crushing；It is further preferred that going out
Bacterium uses irradiation sterilization, in 1000~2,500 1.5~5h of kilorad irradiation sterilization；It is furthermore preferred that the irradiation sterilization time is 3h.
It is preferred according to the present invention, in the step (2), one time fermentation condition are as follows: in 35~38 DEG C of temperature, revolving speed 180
Ferment 24~48h under conditions of~220rpm.
Preferred according to the present invention, in the step (2), one time fermentation media components are as follows, are weight percentage:
1.5~2.5% glucose, 1.0~2.0% peptones, 0.04~0.06% sodium chloride, 0.04~0.06%
The water of beef extract and surplus.
Preferred according to the present invention, in the step (2), the additional amount of Kelp Powder is the 7 of one time fermentation culture medium quality
~12%.
Preferred according to the present invention, in the step (2), the access amount of the bacillus bacterium solution is one time fermentation training
The 1%~5% of matrix amount is supported, the cell concentration of bacillus bacterium solution is 1.5~7.5 × 1010cfu/mL。
It is preferred according to the present invention, in the step (2), removal bacillus thallus using centrifuge separation, 7000~
8~12min is centrifuged under conditions of 10000rpm.
Above-mentioned seawood meal is degraded with a variety of enzymes that bacillus growth generates, and the viscosity of fermented feed liquid gradually increases
Greatly, in order to not influence subsequent fermentation, it is therefore desirable to be separated by solid-liquid separation.
Preferred according to the present invention, in the step (3), the access amount of the lactic acid bacteria is kelp first time fermented liquid
Long-pending 4~6%, the cell concentration of lactic acid bacteria are 2.5~9.5 × 1010cfu/mL。
Preferred according to the present invention, in the step (3), secondary fermentation is that hair is stood under conditions of 28~33 DEG C of temperature
36~72h of ferment.
It is preferred according to the present invention, in the step (4), it is separated by solid-liquid separation to be centrifuged 10 under the conditions of 7000~10000r/min
~20 minutes.
It is preferred according to the present invention, in the step (4), spray drying condition are as follows: 90 DEG C of inlet temperature, outlet temperature 65
DEG C, spray air pressure 0.1Mpa, dry air amount is adjusted to 0.5m3/L；Low temperature drying temperature is 45~65 DEG C.
It is preferred according to the present invention, further include after-ripening step before solid-liquid separation step in the step (4):
Closed standing under the conditions of second of fermentation liquid of kelp is placed in 8~12 DEG C, 15~30d of after-ripening to get.The work of after-ripening
With to make the active constituent in fermentation liquid tend towards stability by low temperature ageing.
It is preferred according to the present invention, in the step (4), crush to pulverize.
Application of the above-mentioned malignant tumour medical food raw material in preparation malignant tumour health food or drug.
Beneficial effect
1, the mode that first passage of the present invention carries out prebiotic microzyme secondary fermentation to kelp prepares the medical food of malignant tumour
Product raw material, this method can discharge nutriment and active constituent in kelp, and not only safe preparation process is reliable, green high-efficient,
The use of chemical reagent strong acid, highly basic is also effectively prevented, exhaust emission is reduced, fermentation liquid contains dietary fiber, reaches no slag
Discharge meets the strategy and policy of environmentally protective and new and old kinetic energy conversion；
2, through the invention the method preparation malignant tumour medical food raw material, containing it is higher with it is antitumor, anti-
The closely related fucoidin of virus, antibacterial anti-inflammatory, antioxidation, polyphenol, fucose content, and by embodiment preparation
Kelp fermentation liquid all has good inhibiting effect to the growth of neuroglial cytoma；Simultaneously the present invention not only it is complete retain and
The active constituent in kelp is released, the fermentation probiotics can produce glutathione, gamma amino butyric acid, extracellular cellulose
Enzyme, pectase isoreactivity ingredient, increase the type and content of active constituent in tunning, it is medical to further improve tumour
The nutrition and curative effect of food formula raw material.
Detailed description of the invention
Fig. 1 is the histogram of polyphenol recovery rate comparison in embodiment 1 and comparative example 1, comparative example 2；
Fig. 2 is the canonical plotting of polyphenol content testing result in kelp first time fermentation liquid in embodiment 1；
Fig. 3 is the inhibition neuroglial cytoma U87/LC3 Electronic Speculum of malignant tumour medical food raw material prepared by embodiment 2
Photo；
Wherein: after a, application when 0h U87/LC3 cell Electron microscope, after b, application when 4h U87/LC3 cell Electronic Speculum
Structure, after c, application when 8h U87/LC3 cell Electron microscope, after d, application when 12h U87/LC3 cell Electron microscope, e,
After application when 16h U87/LC3 cell Electron microscope；
Specific embodiment
Technical solution of the present invention is further elaborated with embodiment with reference to the accompanying drawings of the specification, but the present invention is protected
It is without being limited thereto to protect range.Unless otherwise specified, all technical and scientific terms used herein has and the affiliated technology of the application
The normally understood identical meanings of the those of ordinary skill in field.
Biological material source
The source of heretofore described lactic acid bacteria does not limit, using the lactic acid bacteria of this field food sources；Implement
Lactobacillus plantarum deposit number CICC 20261, Lactobacillus casei deposit number CICC 20277, acidophilus involved in example and comparative example
Lactobacillus deposit number CICC 22150, streptococcus thermophilus deposit number CICC 20370 are purchased from Chinese industrial microorganism fungus kind guarantor
It hides administrative center (CICC)；Lactobacillus gasseri deposit number JCM11657, lactobacillus deposit number JCM2009 is curled, is purchased from Japan
Organism Depositary (Japan collection of microorganisms)；
Heretofore described bacillus is probiotics, and the present invention does not limit the source of the bacillus, adopts
With the bacillus of this field food sources；Bacillus subtilis involved in embodiment and comparative example is purchased from Chinese industrial
Microbiological Culture Collection administrative center (CICC), deposit number CICC24434；Bacillus pumilus deposit number CGMCC 1.7925,
Bacillus licheniformis deposit number CGMCC 1.15832, bacillus coagulans deposit number CGMCC 1.10832 are purchased from Chinese common
Microbiological Culture Collection administrative center；
Test material used in embodiment is the commercially available material of this field routine, can be bought by commercial channel
It arrives.
Crush the atomizer for using Jiangsu Province Ming Feng Machinery Manufacturing Co., Ltd., ordinary commercial products；
Spray drying device is European Union's board OM-1500/OM-1500A lab spray dryer, and the condition of spray drying is set
Be set to: 90 DEG C of inlet temperature, 65 DEG C of outlet temperature, spray air pressure 0.1Mpa, dry air amount is adjusted to 0.5m3/L；Commonly
Commercial product；
It is dry that Medical Equipment Plant of Co., Ltd GZX-9240MBE digital display air dry oven, low temperature are tested using rich prove to be true after interrogation in Shanghai
The temperature of drying is 45~65 DEG C, is dried to water content lower than 3%；Ordinary commercial products；
It pulverizes and same and development in science and technology Co., Ltd the broken machine of KC-701 plant ultra-micropowder, common city is started using Beijing
Sell product；
Mixing is mixed using the V-type mixing machine of Changzhou Qianjiang drying equipment factory production, ordinary commercial products.
Detection method
Fucoidin: according to (fringe light, laminarin isolate and purify and activity research [D] Shandong University, 2004) in
The method of record is detected；
Fucose: according in GB/5009.168-2016 People's Republic of China (PRC) agricultural industry criteria NY/T2279-2202
Method is detected；
Brown algae polyphenols: according to (Yang Huicheng, kelp (Laminaria japonica Aresch) polyphenol extraction separation and
Its antitumor antibacterial activity research [D] Chinese Marine University, 2008) method recorded in is detected；
Polysaccharide: the phend-sulphuric acid that the measurement according to Thick many candies in the outlet SN/T 4260-2015 plant derived foods is recorded
It is detected；
Inhibiting tumour cells experiment: according to (Yang Huicheng, kelp (Laminaria japonica Aresch) polyphenol mention
Take separation and its antitumor antibacterial activity research [D] Chinese Marine University, 2008) in record method detected；
Embodiment 1
A method of malignant tumour medical food raw material is prepared using probiotics fermention kelp, comprising the following steps:
(1) kelp of water content≤5% is crushed to partial size is 500 μm, and Kelp Powder is made in sterilizing；
The kelp is that fresh kelp is dried under the conditions of temperature 70 C after pure water rinses 3 times, is made；
The step of sterilizing, is as follows: in 1500 kilorad irradiation sterilization 3h；
(2) one time fermentation culture medium is added in Kelp Powder made from step (1), the additional amount of Kelp Powder is one time fermentation training
The 10% of matrix amount is supported, is uniformly mixed, after then accessing bacillus bacterium solution, under conditions of 36 DEG C of temperature, revolving speed 200rpm
One time fermentation 36h is carried out, 10min is centrifuged under conditions of 8500rpm and removes bacillus thallus, kelp is made and ferments for the first time
Liquid；
The bacillus is bacillus pumilus；
The one time fermentation media components are as follows, are weight percentage:
1.5% glucose, 1.0% peptone, 0.04% sodium chloride, 0.04% beef extract and the water of surplus；
The access amount of the bacillus bacterium solution is the 3% of one time fermentation culture medium quality, the bacterium of bacillus bacterium solution
Bulk concentration is 5 × 1010cfu/mL；
(3) inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium into kelp first time fermentation liquid made from step (2)
Mixed Microbes, the access amount of the lactic acid bacteria are the 5% of kelp first time fermentating liquid volume, the cell concentration of lactic acid bacteria is 3.0 ×
1010Cfu/mL is left to ferment 36h under conditions of 30 DEG C of temperature, and second of fermentation liquid of kelp is made；
The lactic acid bacteria is lactobacillus plantarum；
(4) by second of fermentation liquid made from step (3) through being centrifuged 15 minutes under the conditions of 8500r/min, supernatant is through spraying
After ultramicro grinding, precipitating dry powder is made, mixing supernatant dry powder and precipitating are dry through low temperature drying in dry obtained supernatant dry powder, precipitating
Powder to get.
The spray drying condition are as follows: 90 DEG C of inlet temperature, 65 DEG C of outlet temperature, spray air pressure 0.1Mpa, dry
Air capacity is adjusted to 0.5m3/L；Low temperature drying temperature is 55 DEG C.
According to the method recorded in (Fan Wenle, comprehensive utilizating research [D] the University Of Science and Technology Of Tianjin of kelp, 2006) to step
Suddenly kelp first time fermentation liquid made from (2) is detected, and the results are shown in Table 1:
Fucoidin content and composition in 1 kelp first time fermentation liquid of table
Embodiment 2
A method of malignant tumour medical food raw material is prepared using probiotics fermention kelp, comprising the following steps:
(1) kelp of water content≤5% is crushed to partial size is 10 μm, and Kelp Powder is made in sterilizing；
The kelp is that the kelp after being soaked is dried under the conditions of temperature 45 C after pure water rinses 5 times, is made；
The process that is soaked is as follows: dry kelp being immersed in the pure water of 20 times of quality, be soaked 7h under conditions of 20 DEG C；
The step of sterilizing, is as follows: in 1000 kilorad irradiation sterilization 5h；
(2) one time fermentation culture medium is added in Kelp Powder made from step (1), the additional amount of Kelp Powder is one time fermentation training
Support matrix amount 7%, be uniformly mixed, then access bacillus bacterium solution after, under conditions of 38 DEG C of temperature, revolving speed 180rpm into
Row one time fermentation 48h is centrifuged 12min under conditions of 7000rpm and removes bacillus thallus, and kelp is made and ferments for the first time
Liquid；
The bacillus is the combination of bacillus pumilus, bacillus coagulans；
The one time fermentation media components are as follows, are weight percentage:
2.5% glucose, 2.0% peptone, 0.06% sodium chloride, 0.06% beef extract and the water of surplus.
The access amount of the bacillus bacterium solution is the 1% of one time fermentation culture medium quality, the bacterium of bacillus bacterium solution
Bulk concentration is 7.5 × 1010cfu/mL。
(3) inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium into kelp first time fermentation liquid made from step (2)
Mixed Microbes, the access amount of the lactic acid bacteria are the 4% of kelp first time fermentating liquid volume, the cell concentration of lactic acid bacteria is 4.5 ×
1010Cfu/mL is left to ferment 48h under conditions of 33 DEG C of temperature, and second of fermentation liquid of kelp is made；
The lactic acid bacteria is lactobacillus plantarum, curls the combination in lactobacillus；
(4) by second of fermentation liquid made from step (3) through after-ripening, 10 points are then centrifuged under the conditions of 10000r/min
Clock, the spray-dried obtained supernatant dry powder of supernatant, precipitating is through low temperature drying, after ultramicro grinding, precipitating dry powder is made, mixes supernatant
Dry powder and precipitating dry powder to get.
The spray drying condition are as follows: 90 DEG C of inlet temperature, 65 DEG C of outlet temperature, spray air pressure 0.1Mpa, dry
Air capacity is adjusted to 0.5m3/L；Low temperature drying temperature is 65 DEG C.
Steps are as follows for the after-ripening:
Closed standing under the conditions of second of fermentation liquid of kelp is placed in 8 DEG C, after-ripening 30d to get.The effect of after-ripening is to pass through
Low temperature ageing makes the active constituent in fermentation liquid tend towards stability.
Embodiment 3
A method of malignant tumour medical food raw material is prepared using probiotics fermention kelp, comprising the following steps:
(1) kelp of water content≤5% is crushed to partial size is 1000 μm, and Kelp Powder is made in sterilizing；
The kelp is that the kelp after being soaked is dried under the conditions of 90 DEG C of temperature after pure water rinses 2 times, is made；
The process that is soaked is as follows: dry kelp being immersed in the pure water of 10 times of quality, be soaked 2h under conditions of 40 DEG C；
The step of sterilizing, is as follows: in 2500 kilorad irradiation sterilization 1.5h；
(2) one time fermentation culture medium is added in Kelp Powder made from step (1), the additional amount of Kelp Powder is one time fermentation training
The 12% of matrix amount is supported, is uniformly mixed, after then accessing bacillus bacterium solution, under conditions of 35 DEG C of temperature, revolving speed 220rpm
It carries out one time fermentation for 24 hours, 8min is centrifuged under conditions of 10000rpm and removes bacillus thallus, kelp is made and ferments for the first time
Liquid；
The bacillus be bacillus subtilis, bacillus pumilus, bacillus licheniformis, bacillus coagulans group
It closes；
The one time fermentation media components are as follows, are weight percentage:
2.0% glucose, 1.5% peptone, 0.046% sodium chloride, 0.05% beef extract and the water of surplus.
The access amount of the bacillus bacterium solution is the 5% of one time fermentation culture medium quality, the bacterium of bacillus bacterium solution
Bulk concentration is 1.5 × 1010cfu/mL。
(3) inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium into kelp first time fermentation liquid made from step (2)
Mixed Microbes, the access amount of the lactic acid bacteria are the 6% of kelp first time fermentating liquid volume, the cell concentration of lactic acid bacteria is 5.0 ×
1010Cfu/mL is left to ferment 52h under conditions of 28~33 DEG C of temperature, and second of fermentation liquid of kelp is made；
The lactic acid bacteria be lactobacillus plantarum, Lactobacillus casei, streptococcus thermophilus, lactobacillus gasseri, lactobacillus acidophilus and
Curl the combination of lactobacillus；
(4) by second of fermentation liquid made from step (3) through after-ripening, 10 points are then centrifuged under the conditions of 10000r/min
Clock, the spray-dried obtained supernatant dry powder of supernatant, precipitating is through low temperature drying, after ultramicro grinding, precipitating dry powder is made, mixes supernatant
Dry powder and precipitating dry powder to get.
The spray drying condition are as follows: 90 DEG C of inlet temperature, 65 DEG C of outlet temperature, spray air pressure 0.1Mpa, dry
Air capacity is adjusted to 0.5m3/L；Low temperature drying temperature is 65 DEG C.
Steps are as follows for the after-ripening:
Closed standing under the conditions of second of fermentation liquid of kelp is placed in 8 DEG C, after-ripening 30d to get.The effect of after-ripening is to pass through
Low temperature ageing makes the active constituent in fermentation liquid tend towards stability.
Comparative example 1
The method that water proposes, acid refers to enzymolysis and extraction is respectively adopted to extract fucoidin；According to (Fan Wenle, kelp
Comprehensive utilizating research [D] University Of Science and Technology Of Tianjin, 2006) in record method extract.
Comparative example 2
Method as described in Example 2, the difference is that, one time fermentation uses lactobacillus-fermented, and secondary fermentation uses
Bacillus.
Experimental example 1
According to the method recorded in (Fan Wenle, comprehensive utilizating research [D] the University Of Science and Technology Of Tianjin of kelp, 2006) to reality
Apply in example 1 and comparative example 1 it is obtained difference products heteroglycan and fucoidin content detected, the results are shown in Table 1:
Heteroglycan and fucoidin content balance result in 2 embodiment 1 of table and comparative example 1
As can be seen from Table 2, the polysaccharide of embodiment 1 and fucoidin content with the hot water water in comparative example 1 mentions, acid refers to
Enzymolysis and extraction significantly increases, and makees wherein being reported with immunoregulation effect, antitumor action, antivirus action, antibacterial anti-inflammatory
With, antioxidation, anticoagulant rouge effect, hypoglycemic effect fucoidin content be acidolysis extract 1.93 times, be hot water leaching
1.75 times mentioned；
According to the method recorded in (Fan Wenle, comprehensive utilizating research [D] the University Of Science and Technology Of Tianjin of kelp, 2006) to reality
The content of seeweed polyphenol for applying difference products obtained in example 1 and comparative example 1 is detected, as a result as shown in Figure 1；
As seen from Figure 1, the seeweed polyphenol content with clear anti-tumor activity in embodiment 2 is in comparative example 1
Mention 2.36 times of hot water water, extract 2.18 times, 1.65 times of comparative example 2 of acidolysis.By above data as can be seen that different benefits
The enzymatic hydrolysis sequence of raw bacterium also has significant impact to seeweed polyphenol content.
Experimental example 2
LC3 albumen is a kind of microtubule associated protein, and the variation of specificity, surrounding appearance point can occurs in it in cell autophagy
The structure of poly- shape.And the LC3 albumen of neuroglial cytoma U87/LC3 tumor strain is marked by GFP green fluorescence, can pass through fluorescence
Micro- sem observation.
Influence of the product to neuroglial cytoma U87/LC3 in embodiment 3 is examined with the following method, and step is such as
Under:
(1) neuroglial cytoma U87/LC3 plating cells carry out adherent training in the small glass culture dish of 5cm diameter
It supports, the cell liquid in each culture dish keeps 2mL, incubator culture 18h.
(2) 2.5 ‰ (mass ratio) embodiment, 3 fermentation liquid is added, does 5 groups in parallel respectively, wherein one group is not add implementation
4h, 8h, 12h are cultivated in 3 blank control of example, after 16h, carry out observation variation with fluorescence microscope.
As seen from Figure 3, after product made from embodiment 3 is added in neuroglial cytoma U87/LC3, pass through fluorescence
Micro- sem observation, it can clearly be seen that the poly- shape structure of point occurs in cell peripheral.Specifically, GFP-LC3 albumen is being implemented
Obviously changed after the effect of product made from example 3, wherein the structure of the poly- shape of appearance point is such as around U87/LC3 after effect 4h
Shown in Fig. 3 b, and in subsequent 8h to 12h, the structure of this poly- shape of point is always existed, and gradually increases, to the end always
16h, it can be found that eucaryotic cell structure has ruptured, death of neoplastic cells.This change procedure clearly illustrates that embodiment 3 is made
Autophagy phenomenon occurs for the product meeting inducing cell obtained, life of the kelp fermentation liquid that both prepared by embodiment 3 to neuroglial cytoma
It is long that there is good inhibiting effect.
Claims (10)
1. it is a kind of using probiotics fermention kelp preparation malignant tumour medical food raw material method, which is characterized in that including with
Lower step:
(1) kelp is crushed, Kelp Powder is made；
(2) one time fermentation culture medium is added in Kelp Powder made from step (1), be uniformly mixed, then access bacillus bacterium solution
Afterwards, one time fermentation removes bacillus thallus, and kelp first time fermentation liquid is made；
The bacillus is one of bacillus subtilis, bacillus pumilus, bacillus licheniformis, bacillus coagulans
Or two or more combination；
(3) into kelp first time fermentation liquid made from step (2) inoculating lactic acid bacterium or lactic acid bacteria and Bifidobacterium mixing
Second of fermentation liquid of kelp is made in bacterium, secondary fermentation；
The lactic acid bacteria is lactobacillus plantarum, Lactobacillus casei, streptococcus thermophilus, lactobacillus gasseri, lactobacillus acidophilus and curls
The combination of one or more of lactobacillus；
(4) by second of fermentation liquid made from step (3) through being separated by solid-liquid separation, the spray-dried obtained supernatant dry powder of supernatant, precipitating
Through low temperature drying, after crushing, be made precipitating dry powder, mixing supernatant dry powder and precipitating dry powder to get.
2. the method as described in claim 1, which is characterized in that in the step (1), kelp is for fresh kelp or after being soaked
Kelp, it is dry to water content≤5% after desalination；
It is further preferred that the desalination is to be rinsed 2~5 times using pure water；
It is further preferred that the process that is soaked is as follows: dry kelp being immersed in the pure water of 10~20 times of quality, 20~40
Be soaked 2~7h under conditions of DEG C；
It is further preferred that it is described it is dry use drying mode, 45~90 DEG C of drying temperature.
3. the method as described in claim 1, which is characterized in that in the step (1), powder particle diameter is 10~1000 μm；
Preferably, in the step (1), further include the steps that sterilizing after crushing；It is further preferred that sterilizing is gone out using irradiation
Bacterium, in 1000~2,500 1.5~5h of kilorad irradiation sterilization；It is furthermore preferred that the irradiation sterilization time is 3h.
4. the method as described in claim 1, which is characterized in that in the step (2), one time fermentation condition are as follows: in temperature 35
~38 DEG C, ferment under conditions of 180~220rpm of revolving speed 24~48h；
Preferably, in the step (2), one time fermentation media components are as follows, are weight percentage:
1.5~2.5% glucose, 1.0~2.0% peptones, 0.04~0.06% sodium chloride, 0.04~0.06% beef
The water of cream and surplus.
5. the method as described in claim 1, which is characterized in that in the step (2), the additional amount of Kelp Powder is one time fermentation
The 7~12% of culture medium quality；
Preferably, in the step (2), the access amount of the bacillus bacterium solution is the 1% of one time fermentation culture medium quality
~5%, the cell concentration of bacillus bacterium solution is 1.5~7.5 × 1010cfu/mL。
6. the method as described in claim 1, which is characterized in that in the step (2), removal bacillus thallus is using centrifugation
Separation is centrifuged 8~12min under conditions of 7000~10000rpm.
7. the method as described in claim 1, which is characterized in that in the step (3), the access amount of the lactic acid bacteria is kelp
The 4~6% of first time fermentating liquid volume, the cell concentration of lactic acid bacteria are 2.5~9.5 × 1010cfu/mL；
Preferably, in the step (3), secondary fermentation is that 36~72h is left to ferment under conditions of 28~33 DEG C of temperature.
8. the method as described in claim 1, which is characterized in that in the step (4), be separated by solid-liquid separation as 7000~10000r/
It is centrifuged 10~20 minutes under the conditions of min；
Preferably, in the step (4), spray drying condition are as follows: 90 DEG C of inlet temperature, 65 DEG C of outlet temperature, spray air pressure
Power 0.1Mpa, dry air amount are adjusted to 0.5m3/L；Low temperature drying temperature is 45~65 DEG C.
9. the method as described in claim 1, which is characterized in that in the step (4), before solid-liquid separation step, after further including
Ripe step:
Closed standing under the conditions of second of fermentation liquid of kelp is placed in 8~12 DEG C, 15~30d of after-ripening to get.The effect of after-ripening is
The active constituent in fermentation liquid is set to tend towards stability by low temperature ageing；
Preferably, it in the step (4), crushes to pulverize.
10. malignant tumour medical food raw material made from claim 1 the method is in preparation malignant tumour health food or medicine
Application in object.
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| CN201811103176.4A CN109288042A (en) | 2018-09-20 | 2018-09-20 | A method and application of using probiotics to ferment kelp to prepare medical food raw materials for malignant tumors |
| BE20195597A BE1026585B1 (en) | 2018-09-20 | 2019-09-10 | PROCESS AND APPLICATION FOR THE PREPARATION OF A MEDICAL FOOD RAW MATERIAL FOR MALIGNANT TUMORS OF FERMENTED VARECH USING PROBIOTICS |
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