CN102191189A - Novel probiotic strain and application thereof - Google Patents
Novel probiotic strain and application thereof Download PDFInfo
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- CN102191189A CN102191189A CN2010101226505A CN201010122650A CN102191189A CN 102191189 A CN102191189 A CN 102191189A CN 2010101226505 A CN2010101226505 A CN 2010101226505A CN 201010122650 A CN201010122650 A CN 201010122650A CN 102191189 A CN102191189 A CN 102191189A
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Abstract
The combination of probiotics and food is helpful for inhibiting the growth of pathogens and absorbing special nutritions. However, the probiotics need to survive at high temperature in the process of food processing, and after the probiotics enter the body, the probiotics must go through the test of stomach acid and bile salt to reach the intestines. Accordingly, the invention provides a bacillus subtilis strain BS139, which is characterized by the survivability of resisting environmental stresses, including the characteristics such as acid resistant, bile salt resistant, high temperature resistance and the like. The probiotic strain is preserved in Agricultural Research Service, NRRL and has a preservation number of NRRL No.B-50347, with further applications in the preparation of food commodities, pharmaceutical compositions, and daily necessities.
Description
Technical field
The invention relates to a kind of probiotic bacterium (BS139) of novelty, particularly about a kind of Bacillus subtilus bacterial strain with the tolerance that can resist environmental stress.
Background technology
In human intestinal, have that kind is about more than 400 kinds, the bacterium of sum about 100,000,000 (bacterium in the intestines), wherein 80-85% is a normal microflora, 15-20% is harmful bacterium.Therefore, it is extremely important to keep the flora eubiosis, and its effect comprises that can help swill to decompose digests, and can also make vitamins B group and K, and probiotic bacterium produces lactic acid and acetic acid, can keep the enteron aisle subacidity, and harmful bacterium can't be survived.And the modern is because of material abundance, the diet aspect is more and more exquisiter, exquisite, add aged growth, intestinal bacterium is produced greatly to change, probiotics reduces gradually, harmful bacterium rolls up, and then causes phenomenons such as tired, anxiety, pachylosis, can influence normal absorption of nutrient what is more and produces disease.
So-called probiotic bacterium (Probiotics), the someone is translated into the protista element, gives birth to rhzomorph, bacteria-promoting agent or primary health bacterial classification.According to the definition of World Health Organization (WHO), may be defined as " an amount of give host, make it can produce the viable bacteria of health effect " " probiotic bacterium ".Also can broadly be interpreted as: be applied to the mankind or other animal, balance each other, be of value to host's viable bacteria by improving microorganism in the intestines.Its effect comprises the balance that can improve microorganism in host's intestines, adjusts the composition of intestinal colony, and then can help the human various pathogenic infections of opposing and suppress harmful bacterium, and can improve urogenital tract flora, preventing infection.
Probiotic bacterium is of a great variety, comprise Bacillus subtilus (Bacillus subtilis), Lactobacillus acidophilus (Lactobacillus acidophilus), bulgaricus ccm (Lactobacillusbulgaricus), two qi bacillus bifiduses (Bifidobacterium sp.), dragon root fungus (Bifidobacterium Longum), thermophilic suis (Streptococcusthermophilus) etc., common on the market probiotic bacterium includes yeast and milk-acid bacteria etc., and the product that utilizes milk-acid bacteria to produce is numerous, wherein with fermented-milk, lactobacillus drink, sour milk, yogurt, play department, gastrointestinal drug, pickles, candy, related productss such as biscuit are in the majority, generally speaking, commercially available product focuses mostly in the healthcare product of cultured milk prod and lozenge capsule pattern, it is living environment because of its characteristic conforms milk-acid bacteria, make the milk-acid bacteria can be, and general milk-acid bacteria be at non-dairy product such as candy at the milk-product length of surviving, then be difficult for survival in the jelly.
In addition, the general equal powerful metabolize sugars of milk-acid bacteria, and can produce lactic acid, make product have tart flavour, therefore the product taste is limited to, therefore the commercially available prod is added the numerous food product additive usually at present, as materials such as sucrose, fructose, against the principle of health-preserving, do not limited in order to make mouthfeel, the product that pharmaceutically dosage form is also arranged on the market, but this type of product but embarrass children to accept.
No matter product form why, milk-acid bacteria will be brought into play effect and is necessary for viable bacteria, and the hydrochloric acid in gastric juice value of human body is between pH value 2～3, and many lactic acid bacteria culturers can't be survived under this sour environment, and lactic acid bacteria culturers that therefore can real generation effect in human body is very rare; Simultaneously, the cholate in the intestines also can produce destruction to milk-acid bacteria, and it is very limited to arrive at the milk-acid bacteria of colon by digestive tube.
Moreover the milk-acid bacteria culture condition must strict keyholed back plate, must be under specified temp milk-acid bacteria could well-grown, and milk-acid bacteria non-refractory, finished product can't utilize High Temperature Sterilization, and product can't be put for a long time, easily grows assorted bacterium.
Bacterium can exist with the Endospore kenel under varying environment pressure, to increase the tolerance to environment.Endospore, claim gemma (endospore), statospore again, it is the bacterium of some special population, it mainly is bacteriogenic special hypopus among bacillus Bacillus and the fusobacterium Clostridium, extremely strong resistance is arranged, to heat, alkali, acid, height ooze, organic solvent (for example alcohol), household cleaners and radiation all have strong tolerance.International periodical JOURNAL OF BACTERIOLOGY, p.2692-2700 Apr.2006 confirms that Bacillus subtilis can sprout in enteron aisle, and can form spore once more by sprouting attitude.
This case contriver is at the deficiency in the known technology, through concentrated test and research, and in line with the spirit of working with perseverance, visualize this case " novel probiotic strain and application thereof " eventually, can overcome the deficiency of prior art, a kind of Bacillus subtilus (Bacillus subtilis) bacterial strain BS 139 with the tolerance that can resist environmental stress is provided, comprise acidproof, bile tolerance and characteristic such as high temperature resistant, it is stored in american agriculture research (the Agriculture Agricultural Research Service of DSMZ, NRRL), deposit number is NRRL NO.B-50347.And can further be applied to the preparation aspect of foods prods and medical component.Below for the brief description of this case.
Summary of the invention
Recently the common people value for diet health very much, and biotechnology promotes gradually, therefore begin one's study probiotic bacterium and food are combined, because what probiotic bacterium helped to suppress the pathogeny bacterium in human body multiplies and absorbs special nutrition, therefore increase the bacterium number of probiotic bacterium at human body, help the health of human body.But the bacterium that enters human body via diet, no matter be probiotic bacterium or pathogeny bacterium, all must be by the test of hydrochloric acid in gastric juice and cholate, could arrive enteron aisle smoothly, probiotic bacterium is in the process of food-processing, and high temperature also is a test, therefore how to make the probiotic bacterium both can acidproof, bile tolerance, high temperature resistant, but long-term surviving is in non-dairy product and do not produce acid again, meet product processing procedure, reduce cost of manufacture, will be a very important target.
Bacterial strain of the present invention is to be obtained by institute's separation in the soy cheese goods, soy cheese is a kind of bean product of secondary processing, earlier soya bean is ground to form soya-bean milk and adds gypsum, through stirring, solid, be pressed into hard bean curd, after the surface drying, just form soy cheese after the secondary processing; The method of secondary processing has two kinds:
(1) use soya bean song or soya bean rice song: boilings such as soya bean, rice are in but back koji; Mix the bent maturation of soya bean behind the bean curd salt marsh.
(2) inoculation mucormycosis: the bean curd of surface drying inoculation mucormycosis is cultivated into mold bean curd, soak salt solution again, with the food flavouring maturation.
Owing to make the employed mould of soy cheese, must select the artificial culture bacterial classification of domestication for use, this bacterial strain is that a natural bacterial strain and trace are present in the soy cheese goods, after seeing through sampling, separate and analyzing its biological nature, through laboratory qualification is Bacillus subtilus (Bacillussubtilis) strain isolated of a novelty, and be the probiotic bacterium kind of a novelty, so give the probiotic bacterium numbering BS-139 of this novelty.
Therefore, for realizing purpose of the present invention, one aspect of the present invention provides a kind of Bacillus subtilus (Bacillus subtilis) bacterial strain with the tolerance that can resist environmental stress, and its microbial preservation number is: NRRL NO.B-50347; The classification name is: Bacillus subtilis; The preservation time: on February 1st, 2010; Preservation address: No. 1400 outstanding rice lake, independent main road, southwest, Washington D.C. favour Teng mansion, 20250 (Jamie L.Whitten Building 1400Independence Ave., S.W.Washington DC, 20250); Depositary institution: american agriculture research DSMZ (Agriculture Agricultural Research Service, NRRL).
The morphological feature of Bacillus subtilus bacterial strain of the present invention: thalline with the line separate mode, is inoculated in the Luria-Bertin Agar Plating, cultivates through 37 ℃ of grow aerobicallies overnight.The positive bacterium G of gramstaining (+) is the aerobic bacterium, can produce Endospore, and it is shaft-like that the thalline when the growth logarithmic phase is 4 μ mx1 μ m, has all flagellums, high motility.When nutritious Luria-Bertini (LB) Agar Plating was grown, colony shape was circular, shallow oyster white, and the surface is projection a little, and periphery of bacterial colonies is level and smooth
Another object of the present invention provides a kind of foods prods, and comprising described Bacillus subtilus is probiotic bacterium BS139.
The present invention's another purpose promptly provides a kind of medical component, and comprising described Bacillus subtilus is probiotic bacterium BS139.
The objective of the invention is for the solution problems of the prior art provide a kind of Bacillus subtilus (Bacillus subtilis) bacterial strain is probiotic bacterium BS139, it is stored in U.S. Agricultural Research Service (NRRL), deposit number is NRRL NO.B-50347, Bacillus subtilus bacterial strain of the present invention (being probiotic bacterium BS139) has the tolerance that can resist environmental stress, and it is implemented kenel and comprises bacterium liquid or bacterium powder kenel.The tolerance of probiotic bacterium BS 139 of the present invention comprises that bile tolerance, temperature and sour environment etc. are similar to the condition of human gastrointestinal tract.
The present invention cultivates the probiotic bacterium BS139 with spore, and utilize the control of the dissolved oxygen factor, substratum concentration control techniques to promote its spore production rate, and the reinforcement spore is to the resistance of 85 ℃～90 ℃ of high temperature, make the probiotic bacterium BS139 can acidproof, bile tolerance, and can be high temperature resistant, meet the processing procedure of product, reduce cost of manufacture, turn out do not produce acid probiotic bacterium BS139, but also long-term surviving in non-dairy product.
Simultaneously, also probiotic bacterium BS139 of the present invention can be applied to prepare foods prods, foods prods comprises one or more in drink, food, animal-derived food product, the food additives.
Drink is for example: fermented-milk, lactobacillus drink, cow's milk, milky-drinks, soymilk, sour milk, yogurt, beverage, fruit juice, soya-bean milk, tea bag, tealeaves, coffee powder, cocoa powder, milk powder, bean powder, flour, mineral water, bottled water and chickens' extract.
Food is for example: Tofu pudding, cheese, cheese, freezing yogurt, pudding, tea freeze, celestial careless Ice, candy, suck ingot, Western-style pastry, cake, bread, biscuit, nut, steamed dumping, instant noodles, noodles, upper thread, Green bean noodle, biscuit, ice-creams, soft sweets, chewing gum, cereal food and cure raw material.
Animal-derived food product is for example: poultry and livestock feed, fish meal, feed, feed for pet add and pet supplies.
Food additives is for example: middle cartridge bag, seasoning bag, seasonings, spice, food ingredients, hot sesame oil, sesame oil, sweet oil, hot sauce, chutney, sugar, salt, soy sauce, foodstuff additive, drinking water additive, Ice interpolation, food ingredients, natural matter and healthy raw material.
Simultaneously, probiotic bacterium BS139 of the present invention also can be applicable to prepare medical component, acting in conjunction with probiotic bacterium BS139 and magnesium oxide MgO is an example: magnesium oxide MgO can be used for treating pyrosis, hydrochloric acid in gastric juice and acid dyspepsia aspect medical, also can be used as fugitive laxative (soft stool agent).The resistance of probiotic bacterium BS139 spore of the present invention makes it can enter enteron aisle easily, sprouts the back and improve intestinal microflora in enteron aisle, and the drainage situation is recovered.Wherein said medical component can be made into formulations such as solution, emulsion, powder, lozenge or capsule.
In addition, probiotic bacterium BS139 of the present invention also can be applicable to prepare articles for daily use, and articles for daily use comprise: health-care product, cleaning supplies, cosmetic products, daily necessities, agriculture articles for use.
Described health-care product is heath food, protective foods, stomach protective material, hair growing liquid and hair care agent.
Probiotic bacterium BS139 of the present invention adds the antibacterial ability that cleaning supplies can strengthen cleaning supplies, and wherein said cleaning supplies are shampoo, cleansing milk, profit hair-cream, bath oil, soap, perfumed soap, washing powder, collutory, toothpaste, tooth powder, private parts vaginal washing fluid, diaper, cotton wool, cotton pad, the powder of bathing, the bag of bathing, powder for dipping feet, bubble pin bag and debris removal agent.
Probiotic bacterium BS139 of the present invention adds cosmetic products can form protective membrane; have the function of strengthening skin moisture-keeping, antibiotic, metabolism aging cutin, wherein said cosmetic products are face cream, hand lotion, skin cream, perfume, facial mask, essential oil and cosmetic material additive.
Described daily necessities is shoe-pad, perfume compound, ointment, spray, sprays prickly-heat powder, environment spray, toilet reodorant or water tower or the inner interpolation of water dispenser.
Described agriculture articles for use are agricultural micro organism preparation, soil activating agent, microorganism fertilizer feed additives or microbial fertilizer starter.
Bacillus subtilus bacterial strain of the present invention has following advantage:
Via the technology used in the present invention means, probiotic bacterium BS139 of the present invention can be applied to making, processing or the medical preparation aspect of foods prods.Utilize probiotic bacterium strain isolated of the present invention to have characteristic, under the high temperature that can in the process of food-processing, may meet with, still keep its activity cholate, temperature and sour environment tolerance.Also can be in the human body intestines and stomach under the conditions such as acidity, cholate, survival arrives enteron aisle smoothly.To improve the balance of microorganism in host's intestines, adjust the composition of intestinal microflora, and then can help effects such as human various pathogenic infections of opposing and the harmful bacterium of inhibition.
Description of drawings
Figure 1A: the optical microscopy camera when probiotic bacterium BS139 logarithmic phase of the present invention is grown;
Figure 1B: the colonial morphology of probiotic bacterium BS139 of the present invention;
Fig. 2: the growth curve of probiotic bacterium BS139 of the present invention;
Fig. 3 A～3B: the class source relational tree of probiotic bacterium BS139 of the present invention;
Fig. 4: probiotic bacterium BS139 of the present invention is to the tolerance test result of acid;
Fig. 5: probiotic bacterium BS139 of the present invention is to the tolerance test result of temperature;
Fig. 6: probiotic bacterium BS139 bacterium powder of the present invention is to the tolerance test of cholate;
Fig. 7: probiotic bacterium BS139 bacterium powder of the present invention is to the tolerance test of temperature;
Fig. 8: probiotic bacterium BS139 bacterium powder of the present invention is to the tolerance test of acid;
Fig. 9 A: probiotic bacterium BS139 of the present invention is to the tolerance test of temperature in jelly foodstuff;
Fig. 9 B: the growing state of probiotic bacterium BS139 of the present invention in jelly foodstuff.
Embodiment
Further describe the present invention below in conjunction with specific embodiments and the drawings, advantage of the present invention and characteristics will be more clear along with description.
Morphological feature
Consult Figure 1A, show the light micrograph when probiotic bacterium BS139 logarithmic phase of the present invention is grown.Thalline is suspended in the glycerine, hanging drop after mounting on the slide, with handstand opticmicroscope Leica DMI 3000, object lens are 100 */NA 1.25 oilimmersion, cooperate Photometrics CoolSNAP EZ CCD numeral acquisition thalline image.
Figure 1B shows the colonial morphology of probiotic bacterium BS139 of the present invention.Thalline with the line separate mode, is inoculated in the Luria-Bertin Agar Plating, cultivates through 37 ℃ of grow aerobicallies overnight.Its morphological feature comprises: the positive bacterium G of gramstaining (+), be the aerobic bacterium, and can produce Endospore, when the growth logarithmic phase, thalline is 4 μ mx1 μ m shaft-like (referring to Figure 1A), has all flagellums, high motility.When nutritious Luria-Bertini (LB) Agar Plating was grown, colony shape was circular, shallow oyster white, and the surface is projection a little, periphery of bacterial colonies level and smooth (consulting Figure 1B).
Cultivate properties and characteristics
Fig. 2 shows the growth curve of probiotic bacterium BS139 of the present invention.Thalline of the present invention is inoculated in the liquid substratum of Luria-Bertin, cultivates, write down its bacterium liquid turbidity (Optical density at 600nm) through 37 ℃ of grow aerobicallies.
Physiological property
In addition, probiotic bacterium BS139 of the present invention has following enzyme activity: catalase (catalase), oxydase (oxidase), amylolytic enzyme (α-amylase), protein decomposition enzyme (protease), cellulase lytic enzyme (cellulase), Nattokinase (nattokinase).
Base sequence is analyzed
The karyomit(e) that utilizes probiotic bacterium BS139 of the present invention to extract carries out the PCR sequencing at 16S rDNA and gyrase B gene (gyrB) fragment, and the nucleotide sequence of the 16SrDNA of Bacillus subtilis BS139 bacterial strain, the nucleotide sequence of gyrase B gene are seen sequence table.The result shows that Bacillus subtilus bacterial strain of the present invention belongs to Bacillus subtilis gang, and is close but different with laboratory standard bacterial strain B.subtilis strain 168 (ATCC:23857).Utilize sequence alignment tools (BLAST) to analyse and compare at the database of American National biotechnology information center (NCBI), and the class source relational tree of drawing 16S rDNA and gyraseB gene (is consulted Fig. 3 A～3B), the result shows the Bacillus subtilis bacterial strain that this bacterial strain can be classified as a novelty, and called after probiotic bacterium BS139.
Embodiment 1: the fermentation culture of probiotic bacterium BS139
Fermentation culture process in, its emphasis step comprises: (one) probiotic bacterium screening: during fermentation with carbon source: glucose (Glucose), nitrogenous source: biochemical reagents such as soybean protein and NaCl are cooked strain fermentation, and in centrifuge tube with shake a bottle processing; (2) aerobic product spore probiotic's culture: insert the 5L tank culture, after insert the 100L tank culture again, nutrient solution leaves standstill oxygen deprivation and kept 2 afterwards, increases the microorganism spore production rate; (3) dominant growth triage techniques: when cultivating probiotic bacterium BS 139, per hour get bacterium liquid one time, note down its growth efficiency, calculate the highest time point of its growth efficiency, grow mid-term (mid-log phase) on 6 hours achievable pair numbers.
Embodiment 2: probiotic bacterium BS139 spore heatproof, productive rate lift technique
Promoting spore heatproof and yield aspects, utilize the control of the dissolved oxygen factor: improve dissolved oxygen with a large amount of aerations, oxygen requirement with bacterium in the supply nutrient solution, if reduce the dissolved oxygen of nutrient solution, environmental change will make the production by biological spore, can improve spore production rate, and utilize the control of substratum concentration: utilize sterilized water with volume ratio dilution nutrient solution concentration, utilize concentration control obtain high yield spore speed optimum medium concentration (with the dilution nutrient solution be original content 50% and 30%), different concns is measurement spore productive rate down.Find that by difference test above technology can promote spore production rate, spore heatproof and the productive rate of probiotic bacterium, strengthen spore to the resistance of 85 ℃～90 ℃ of high temperature and improve its spore production rate.
Embodiment 3: probiotic bacterium BS139 is to the tolerance test of acid
The preceding preparation of sample
At first a little bacterium powder is steeped in nutrient solution, activate to be coated in after four hours to be placed on the substratum and carry out cultivation overnight (overnight) in 37 ℃ of incubators (incubator), and do the same manner preparation with enterohemorrhagic Escherichia coli (EHEC).
After finishing the sample preparation, carry out according to the following step:
1. a little adds in the 6ml nutrient solution of centrifuge tube dress with the bacterium colony on the toothpick picking square position, is placed in 37 ℃ of incubators (incubator) rotating and culturing 16 hours.
2. the bacterium liquid branch of cultivating being installed to aseptic round bottom culture tube, is respectively 1-0,1-1,1-2,1-3 and 1-1s, 1-2s, 1-3s totally 7 pipes, every pipe 0.8ml.
Above-mentioned 7 pipes are centrifugal with 7000rpm, 2min 3., outwell supernatant liquor, centrifugal with 7000rpm, 10sec rapidly again, outwell supernatant liquor again and only stay precipitation (pellets).
4. the PBS (pH 2.0) that 1-1s, 1-2s, 1-3s is added 0.8ml respectively, all the other 4 pipes add the PBS (pH 7.3) of 0.8ml, and pellets is broken up in vibrations (vortex).
5. 1-0 is taken out 20 μ l and do serial dilution (utilizing PBS pH7.3 dilution), get 10
6, 10
7Dilution bacterium liquid 0.1ml utilizes substratum to be coated with dish counting overnight, all the other 6 pipes are all put into 37 ℃ of incubators and are cultivated (150rpm), each hour got paired 2 pipe serial dilutions, the suitable extension rate of getting 0.1ml is coated with dish counting overnight with substratum, get 1-1 and 1-1S dilution in promptly the 1st hour and be coated with dish, the experiment of next carrying out in two hours by that analogy.
6. carry out three in the above described manner and repeat experiment, and the data of record counting gained.
7. get the employing of intestinal bleeding intestinal bacteria (EHEC) bacterium liquid and test with above-mentioned mode, measured data are used as the control group of experiment.
8. this experiment need be carried out pH2.0, pH3.0, pH4.0, pH7.3 totally 4 kinds of acid tolerance tests, therefore must repeat above-mentioned experiment respectively with the PBS of pH3.0, pH4.0.
The result sees also Fig. 4, shows the tolerance test result of BS139 to acid.Experimental observations finds that BS139 can be survived at most 2 hours under pH 4.0 environment.
Embodiment 4: probiotic bacterium BS139 is to the tolerance test of temperature
In the present embodiment, at first carry out as preparation and following steps before the sample of embodiment 3:
1. a little adds in the 6ml nutrient solution of centrifuge tube dress with the bacterium colony on the toothpick picking square position, is placed in 37 ℃ of incubators (incubator) rotating and culturing 16 hours.
2. the bacterium liquid branch that will cultivate installs to aseptic round bottom culture tube, and mark 1-0,1-1,1-2 totally 3 manage every pipe 0.8ml respectively.
Above-mentioned 3 pipes are centrifugal with 7000rpm, 2min 3., outwell the rapid centrifugal 7000rpm of supernatant liquor, 10sec and outwell supernatant liquor again and only stay precipitation (pellets).
4. above-mentioned 3 pipes are added the PBS (pH 7.3) of 0.8ml, pellets is broken up in vibrations.
5. do serial dilution (utilizing PBS pH7.3 dilution) with taking out 20 μ l in the 1-0 pipe, get 10 of 0.1ml
6, 10
7Dilution bacterium liquid carries out substratum and is coated with dish counting overnight.
6. paired 2 pipes (1-1,1-2) being put into 100 ℃ of dried baths cultivates, get 1 pipe bacterium liquid every 15 minutes and carry out serial dilution, and the bacterium liquid of getting the suitable extension rate of 0.1ml carries out substratum and is coated with dish meter book overnight, that is gets 1-1 on the 15th minute, gets the 1-2 dilution and is coated with dish in the 30th minute.
7. do three in the above described manner and repeat experiment, and the data of record counting gained.
8. get the employing of EHEC bacterium liquid and test with above-mentioned mode, measured data are used as the control group of experiment.
9. this experiment temperature test that need carry out has 50 ℃, 75 ℃, 80 ℃, 100 ℃, and the test temperature resistance time has 10 minutes, 15 minutes, 30 minutes, 1 hour.Therefore according to aforesaid way, adjust the temperature and the storage period of dried bath, test and note down data then according to measuring target.
The result consults Fig. 5, shows the tolerance test result of BS139 to temperature.Experimental observations finds that the BS139 nutrient solution then also has 20% survival rate approximately in 50 ℃ of effects in the time of 30 minutes.
Embodiment 5: probiotic bacterium BS139 bacterium powder is to the tolerance test of cholate
The preceding preparation of sample
The bacterium powder that will test is managed the bacterium powder with the first packing 20 of centrifuge tube (eppendorf tubes), and with the sample as experiment usefulness, the about 0.1g of every pipe bacterium grain weight places-20 ℃ of refrigerators.1. from-20 ℃ of refrigerators, get 1 pipe 0.1g bacterium powder, fully shake mixing in the nutrient solution (containing 3% cholate, bile salt) with this bacterium powder adding 10ml.
2. divide the aseptic round bottom culture tube of 4 pipes of packing into, every pipe 1ml is denoted as 1-0,1-1,1-2,1-3 respectively.
3. the 1-0 culture tube is taken out 20 μ l and carry out serial dilution (utilizing PBS pH7.3 dilution), get 10 of 0.1ml
5, 10
6Dilution bacterium liquid carries out substratum and is coated with dish counting overnight, all the other 3 pipes (1-1,1-2,1-3) are put into 37 ℃ of incubator rotating and culturing (150rpm), get 1 pipe every 1 hour and carry out serial dilution, the suitable dilution bacterium liquid of getting 0.1ml carries out substratum and is coated with dish counting overnight, that is got the 1-1 dilution on the 1st hour and be coated with dish, the experiment of next carrying out in two hours by that analogy.
4. carry out three in the above described manner and repeat experiment, and the data of record counting gained.
5. finish after the test of 3% cholate resistance, change the nutrient solution that becomes to contain 0.3% cholate and carry out the resistance test in a manner described, and record gained data are to compare.
The result consults Fig. 6, shows the tolerance test of BS139 bacterium powder to cholate.Experimental result shows that BS 139 containing under the environment of 3% and 0.3% cholate effect 3 hours, also has 20% survival rate nearly.
Embodiment 6: probiotic bacterium BS139 bacterium powder is to the tolerance test of temperature
In the present embodiment, at first carry out: 1. from-20 ℃ of refrigerators, get 1 pipe 0.1g bacterium powder, fully shake mixing in the nutrient solution with this bacterium powder adding 10ml as preparation and following steps before the sample of embodiment 5.
2. divide the aseptic round bottom culture tube of 3 pipes of packing into, every pipe 1ml is denoted as 1-0,1-1,1-2 respectively.
3. the 1-0 test tube is taken out 20 μ l and carry out serial dilution (utilizing PBS pH7.3 dilution), get 10 of 0.1ml
5, 10
6Dilution bacterium liquid carries out substratum and is coated with dish counting overnight, all the other 2 pipes (1-1,1-2) are put in 100 ℃ of dried baths and are cultivated, got 1-1 respectively at the 10th minute and the 30th minute respectively and the 1-2 test tube carries out serial dilution, the suitable dilution bacterium liquid of getting 0.1ml carries out substratum and is coated with dish counting overnight.
4. carry out 3 repeated experiments in the above described manner, and the data of record counting gained.
5. the temperature of this experiment test has 50 ℃, 75 ℃, 80 ℃, 100 ℃ totally 4 kinds, and the temperature that therefore also must change dried bath repeats above-mentioned experiment, to record the survival data of each temperature.
The result consults Fig. 7, shows the tolerance test of BS139 bacterium powder to temperature.Experimental result shows that BS 139 bacterium powder almost do not have the bacterium survival under 100 ℃ temperature, 80% above survival rate is arranged at 75 ℃, 80 ℃ temperature actions in 30 minutes.
Embodiment 7: probiotic bacterium BS139 bacterium powder is to the tolerance test of acid
In the present embodiment, at first carry out: 1. from-20 ℃ of refrigerators, get 1 pipe 0.1g bacterium powder, fully shake mixing among the PBS (pH4.0) with this bacterium powder adding 10ml as preparation and the following step before the sample of embodiment 5.
2. divide the aseptic round bottom culture tube of 4 pipes of packing into, every pipe 1ml is denoted as 1-0,1-1,1-2,1-3 respectively.
3. the 1-0 culture tube is taken out 20 μ l and carry out serial dilution (utilizing PBS pH7.3 dilution), get 10 of 0.1ml
5, 10
6Dilution bacterium liquid carries out substratum and is coated with dish counting overnight, all the other 3 pipes (1-1,1-2,1-3) are put into 37 ℃ of incubator rotating and culturing (150rpm), got 1 pipe every 1 hour and carry out serial dilution, the suitable dilution bacterium liquid of getting 0.1ml carries out substratum and is coated with dish counting overnight.
4. carry out 3 repeated experiments in the above described manner, and the data of record counting gained.
5. this experiment pH that will test has 2.0,3.0,4.0,7.3 totally 4 kinds, and the pH value that therefore also must change PBS repeats above-mentioned experiment method, to record data.
The result consults Fig. 8, shows the tolerance test of BS139 bacterium powder to acid.The BS139 that finds the bacterium powder according to experimental result is significantly better than vegetative state bacterium liquid for the tolerance of acid, and the back survival rate of cultivating 3 hours up to pH3.0 just reduces gradually.
According to above experimental result, BS139 bacterium powder obviously is better than BS139 cultivation bacterium liquid in the tolerance of acid, cholate or temperature, its reason is that the Endospore in the BS139 bacterium powder is quite a lot of, cultivate the content of bacterium liquid miospore far above BS139, and Endospore is the mode that BS139 is survived under severe environment, and it can promote the tolerance of the external surface pressure of BS139.
Embodiment 8: probiotic bacterium BS139 tolerance to temperature in jelly foodstuff is tested
The preceding preparation of sample
The box-packed jelly elder generation branch that contains profitable probliotics BS139 is installed in 2 centrifuge tubes (50ml), fasten the back and seal opening, leave in 4 ℃ of refrigerators with parafilm.1. get a pipe jelly and come out from 4 ℃ of refrigerators, the jelly of getting about 2ml with the soupspoon of sterilizing is respectively packed in the centrifuge tube of 15ml, adds the PBS (pH 7.3) of 1ml respectively, adds granulated glass sphere again and shakes and smash jelly.
2. therefrom get 2 each 2ml of pipe with pipettor (pipet) and insert in the centrifuge tube, indicate 1-0,1-1 respectively.
3. with PBS (pH 7.3) the 1-0 test tube is carried out serial dilution, get 10 of 0.1ml
3, 10
4Dilution bacterium liquid carries out substratum and is coated with dish counting overnight, and institute's value is promptly represented the number of surviving.
4. 90 ℃ of heating of 1-1 test tube same afterwards dilution in 10 minutes is coated with dish counting overnight, institute's value is promptly represented the spore count of surviving.
5. carry out three as stated above and repeat experiment, and record counting gained data.
6. after jelly is stored in 4 ℃ of refrigerator different times, counts in above-mentioned identical experiment mode and to deposit viable count and spore count.
The result consults Fig. 9 A, is presented at the tolerance test to temperature of BS139 in the jelly.Experimental result shows, the BS139 in the jelly is placing 90 ℃ to also have nearly survival rate more than 70% after 10 minutes.
Then consult Fig. 9 B, it shows the growing state of BS139 in jelly.This experimental data is to get the bacterium colony mean number that 1-0 three in the above-mentioned experiment repeats to test the counting gained, and it is converted into colony number contained among the 1ml.The bacterium number that can find the BS139 in the jelly according to the result increased and reduces gradually along with the time.
Pass through present embodiment; can understand probiotic bacterium BS139 can be by the process of various processing; make and form different food or supplementary food; in addition; probiotic bacterium BS 139 of the present invention also can be applicable to prepare articles for daily use, for example: heath food, protective foods, stomach protective material, health-care product, hair growing liquid and hair care agent.
Embodiment 9: food-processing probiotic bacterium BS139 mixes the bacterium technology of preparing
Characteristic based on BS139, be very suitable for making beneficial bacterium jelly, its step comprises that (one) high temperature oozes bacterium and makes: the jelly colloid admixture that melts in heat, adding the pityrosporion ovale powder in 80 ℃ of high temperature stirs, then be cooled to again and carry out pasteurizing in 75 ℃, be cooled to 10 ℃ of condensation moulding again after injecting container thereafter; (2) moulding low temperature treatment technology: the finished product after will lowering the temperature places 10 ℃ to handle 30 minutes, can impel the acidproof spore of activatory vegetative state bacterium continuation formation heatproof in the product this moment.And in the storage time, keep activity stabilized, can reach all interior viable counts greater than 5x10
8Cfu/100ml; After 4 ℃ of low temperature stored for three weeks, viable count was greater than 3x10
8Cfu/100ml.The beneficial bacterium jelly that present embodiment is made, the U.S. check of the platform scientific ﹠ technical corporation that its edible test system breeds center garrison manufacturer by Yangming Univ.'s innovation carries out the soundness test of food microorganisms test and normal food.
By above embodiment as can be known, the utility value on the probiotic bacterium BS139 tool industry provided by the present invention is so the present invention meets the important document of patent.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
Sequence table
＜110〉Lin Xiuxia
＜120〉Xin Ying probiotic strain and application thereof
<160>2
<210>1
<211>1433
<212>DNA
<213>Bacillus subtilis
＜223〉16SrDNA of Bacillus subtilis strain isolated BS139
<400>1
agtcgagcgg acagatggga gcttgctccc tgatgttagc ggcggacggg tgagtaacac 60
gtgggtaacc tgcctgtaag actgggataa ctccgggaaa ccggggctaa taccggatgg 120
ttgtttgaac cgcatggttc aaacataaaa ggtggcttcg gctaccactt acagatggac 180
ccgcggcgca ttagctagtt ggtgaggtaa cggctcacca aggcaacgat gcgtagccga 240
cctgagaggg tgatcggcca cactgggact gagacacggc ccagactcct acgggaggca 300
gcagtaggga atcttccgca atggacgaaa gtctgacgga gcaacgccgc gtgagtgatg 360
aaggttttcg gatcgtaaag ctctgttgtt agggaagaac aagtaccgtt cgaatagggc 420
ggtaccttga cggtacctaa ccagaaagcc acggctaact acgtgccagc agccgcggta 480
atacgtaggt ggcaagcgtt gtccggaatt attgggcgta aagggctcgc aggcggtttc 540
ttaagtctga tgtgaaagcc cccggctcaa ccggggaggg tcattggaaa ctggggaact 600
tgagtgcaga agaggagagt ggaattccac gtgtagcggt gaaatgcgta gagatgtgga 660
ggaacaccag tggcgaaggc gactctctgg tctgtaactg acgctgagga gcgaaagcgt 720
ggggagcgaa caggattaga taccctggta gtccacgccg taaacgatga gtgctaagtg 780
ttagggggtt tccgcccctt agtgctgcag ctaacgcatt aagcactccg cctggggagt 840
acggtcgcaa gactgaaact caaaggaatt gacgggggcc cgcacaagcg gtggagcatg 900
tggtttaatt cgaagcaacg cgaagaacct taccaggtct tgacatcctc tgacaatcct 960
agagatagga cgtccccttc gggggcagag tgacaggtgg tgcatggttg tcgtcagctc 1020
gtgtcgtgag atgttgggtt aagtcccgca acgagcgcaa cccttgatct tagttgccag 1080
cattcagttg ggcactctaa ggtgactgcc ggtgacaaac cggaggaagg tggggatgac 1140
gtcaaatcat catgcccctt atgacctggg ctacacacgt gctacaatgg acagaacaaa 1200
gggcagcgaa accgcgaggt taagccaatc ccacaaatct gttctcagtt cggatcgcag 1260
tctgcaactc gactgcgtga agctggaatc gctagtaatc gcggatcagc atgccgcggt 1320
gaatacgttc ccgggccttg tacacaccgc ccgtcacacc acgagagttt gtaacacccg 1380
aagtcggtga ggtaaccttt taggagccag ccgccgaagg tgggacagat gat 1433
<210>2
<211>1259
<212>DNA
<213>Bacillus subtilis
＜223〉the gyrase B of Bacillus subtilis strain isolated BS139
<400>2
gaagtcatta tgacggtgct tcatgctggt ggtaagtttg acggaagcgg ctataaagta 60
tccggaggat tacacggtgt aggtgcgtcg gtcgtaaacg cactatcaac agagcttgat 120
gtgacggttc accgtgacgg taaaattcac cgccaaacct ataaacgcgg agttccggtt 180
acagaccttg aaatcattgg cgaaacggat catacaggaa cgacgacaca ttttgtcccg 240
gaccctgaaa ttttctcaga aacaaccgag tatgattatg atctgcttgc caaccgcgtg 300
cgtgaattag cctttttaac aaagggcgta aacatcacga ttgaggataa acgtgaagga 360
caagagcgca aaaatgaata ccattacgaa ggcggaatta aaagttatgt agagtattta 420
aaccgctcta aagaggttgt ccatgaagag ccgatttaca ttgaaggcga aaaggacggc 480
attacggttg aagtggcttt gcaatacaat gacagctaca caagcaacat ttactcgttt 540
acaaacaaca ttaatacgta cgaaggcggt acccatgaag ctggcttcaa aacgggcctg 600
actcgtgtta tcaacgatta cgccagaaaa aaagggctta ttaaagaaaa tgatccaaac 660
ctaagcggag atgacgtaag ggaagggctg acagcgatta tttcaatcaa acaccctgat 720
ccgcagtttg agggccaaac gaaaacaaag ctgggcaact cagaagcacg gacgatcacc 780
gatacgttat ttctacggc gatggaaaca tttatgctgg aaaatccaga tgcggccaaa 840
aaaattgtcg ataaaggctt aatggcggca agagcaagaa tggctgcgaa aaaagcccgt 900
gaactaacac gtcgtaagag tgctttggaa atttcaaact tgcccggtaa gttagcggac 960
tgctcttcaa aagatccgag catctccgag ttatatatcg tagagggtga ctctgccgga 1020
ggatctgcta aacaaggacg cgacagacat ttccaagcca ttttgccgct tagaggtaaa 1080
atcctaaacg ttgaaaaggc cagactggat aaaatccttt ctaacaacga agttcgctct 1140
atgatcacag cgctcggcac aggtattggg gaagacttca accttgagaa agcccgttac 1200
cacaaagttg tcattatgac agatgcagac gtaganggcg cgcacatcag aacactgct 1259
Claims (21)
1. a Bacillus subtilus (Bacillus subtilis) bacterial strain, its microbial preservation number are: NRRL NO.B-50347, this bacterial strain has the tolerance that can resist environmental stress.
2. Bacillus subtilus bacterial strain as claimed in claim 1, the enforcement kenel of wherein said Bacillus subtilus bacterial strain is a bacterium liquid.
3. Bacillus subtilus bacterial strain as claimed in claim 1, the enforcement kenel of wherein said Bacillus subtilus bacterial strain is the bacterium powder.
4. Bacillus subtilus bacterial strain as claimed in claim 1, the tolerance that wherein said Bacillus subtilus bacterial strain has is the cholate tolerance.
5. Bacillus subtilus bacterial strain as claimed in claim 1, the tolerance that wherein said Bacillus subtilus bacterial strain has is a temperature tolerance.
6. Bacillus subtilus bacterial strain as claimed in claim 1, the tolerance that wherein said Bacillus subtilus bacterial strain has is sour tolerance.
7. a foods prods comprises edibility material and Bacillus subtilus bacterial strain as claimed in claim 1.
8. foods prods as claimed in claim 7, wherein said foods prods are one or more in drink, food, animal-derived food product, the food additives.
9. foods prods as claimed in claim 8, wherein said drink are one or more in fermented-milk, lactobacillus drink, cow's milk, milky-drinks, soymilk, sour milk, yogurt, beverage, fruit juice, soya-bean milk, tea bag, tealeaves, coffee powder, cocoa powder, milk powder, bean powder, flour, mineral water, bottled water, the chickens' extract.
10. foods prods as claimed in claim 8, wherein said food are that Tofu pudding, cheese, cheese, freezing yogurt, pudding, tea freeze, celestial careless Ice, candy, suck ingot, Western-style pastry, cake, bread, biscuit, nut, steamed dumping, instant noodles, noodles, upper thread, Green bean noodle, biscuit, ice-creams, soft sweets, chewing gum, cereal food, cure in the raw material one or more.
11. foods prods as claimed in claim 8, wherein said animal-derived food product are in poultry and livestock feed, fish meal, feed, feed for pet additive, the pet supplies one or more.
12. foods prods as claimed in claim 8, wherein said food additives are in middle cartridge bag, seasoning bag, seasonings, spice, food ingredients, hot sesame oil, sesame oil, sweet oil, hot sauce, chutney, sugar, salt, soy sauce, foodstuff additive, drinking water additive, Ice interpolation, food ingredients, natural matter, the healthy raw material one or more.
13. a medical component comprises Bacillus subtilus bacterial strain as claimed in claim 1.
14. medical component as claimed in claim 13, the formulation of wherein said medical component are solution, emulsion, powder, lozenge or capsule.
15. articles for daily use comprise Bacillus subtilus bacterial strain as claimed in claim 1.
16. articles for daily use as claimed in claim 15, wherein said articles for daily use are in health-care product, cleaning supplies, cosmetic products, daily necessities, the agriculture articles for use one or more.
17. articles for daily use as claimed in claim 16, wherein said health-care product are in heath food, protective foods, stomach protective material, hair growing liquid, the hair care agent one or more.
18. articles for daily use as claimed in claim 16, wherein said is that cleaning supplies are one or more in shampoo, cleansing milk, profit hair-cream, bath oil, soap, perfumed soap, washing powder, collutory, toothpaste, tooth powder, private parts vaginal washing fluid, diaper, cotton wool, cotton pad, the powder of bathing, the bag of bathing, powder for dipping feet, bubble pin bag, the debris removal agent.
19. articles for daily use as claimed in claim 16, wherein said cosmetic products are in face cream, hand lotion, skin cream, perfume, facial mask, essential oil, the cosmetic material additive one or more.
20. articles for daily use as claimed in claim 16, wherein said daily necessities are in shoe-pad, perfume compound, ointment, spray, sprays, prickly-heat powder, environment spray, toilet reodorant or water tower, the water dispenser internal additives one or more.
21. articles for daily use as claimed in claim 16, wherein said agriculture articles for use are one or more in agricultural micro organism preparation, soil activating agent, microorganism fertilizer feed additives, the microbial fertilizer starter.
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| CN103564345A (en) * | 2013-11-19 | 2014-02-12 | 绿雪生物工程(深圳)有限公司 | Semi-fermented acidic milk pudding and preparation method thereof |
| CN103614327A (en) * | 2013-11-27 | 2014-03-05 | 北京昕大洋科技发展有限公司 | Bacillus subtilis and use thereof |
| US9205116B2 (en) | 2011-04-29 | 2015-12-08 | Auburn University | Bacillus bacteria for use in treating and preventing infection in aquatic animals |
| CN105441486A (en) * | 2015-12-15 | 2016-03-30 | 贵州大学 | Method for producing foot bath powder from waste tea slag through Eurotium amstelodami |
| CN105661013A (en) * | 2016-03-09 | 2016-06-15 | 广东海洋大学 | Method for preparing compound type growth-promoting feed additive by fermenting Chinese herbal medicines |
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| CN108603161A (en) * | 2015-11-09 | 2018-09-28 | 中国科学院遗传与发育生物学研究所 | Bacillus strain with beneficial activity and preparation |
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| US9205116B2 (en) | 2011-04-29 | 2015-12-08 | Auburn University | Bacillus bacteria for use in treating and preventing infection in aquatic animals |
| US9603879B2 (en) | 2011-04-29 | 2017-03-28 | Auburn University | Bacillus bacteria for use in treating and preventing infection in aquatic animals |
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| CN105661013A (en) * | 2016-03-09 | 2016-06-15 | 广东海洋大学 | Method for preparing compound type growth-promoting feed additive by fermenting Chinese herbal medicines |
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| CN107302997A (en) * | 2016-04-19 | 2017-10-31 | 林秀霞 | A kind of composition for suppressing lipopexia and application thereof |
| CN105944130A (en) * | 2016-05-24 | 2016-09-21 | 山东省科学院生物研究所 | Odor-resistant and antibacterial pad attached with probiotics and production method and application of odor-resistant and antibacterial pad |
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