CN104630099A - Bifidobacterium longum strain and application thereof in preparing active Bifidobacterium fermented beverage - Google Patents

Bifidobacterium longum strain and application thereof in preparing active Bifidobacterium fermented beverage Download PDF

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CN104630099A
CN104630099A CN201510023615.0A CN201510023615A CN104630099A CN 104630099 A CN104630099 A CN 104630099A CN 201510023615 A CN201510023615 A CN 201510023615A CN 104630099 A CN104630099 A CN 104630099A
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bifidobacterium
juice
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刘国荣
苏航
宋振芹
王成涛
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Beijing Technology and Business University
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Abstract

The invention discloses a Bifidobacterium longum strain and application thereof in preparing an active Bifidobacterium fermented beverage. The collection number of the Bifidobacterium longum BF-10 is CGMCC No.7789. The Bifidobacterium longum BF-10 CGMCC No.7789 can be used for preparing an active Bifidobacterium fermented beverage. The prepared Bifidobacterium fermented sea buckthorn/Chinese wolfberry composite viable organism beverage has the advantages of mild color, favorable mouthfeel, favorable flavor, high viable count and long shelf life, can display the nutrient and physiological effects of the sea buckthorn and Chinese wolfberry, can enhance the health-care action of the Bifidobacterium, and is a health-care beverage with multiple physiological functions.

Description

One strain bifidus longum bb and preparing the application in living bifidobacteria fermented drink
Technical field
The present invention relates to a strain bifidus bacillus and preparing the application in living bifidobacteria fermented drink, particularly the technique of a strain bifidus longum bb bacterial strain and fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage thereof.
Background technology
Lactobacillus drink, the nutrition abundant due to it and health-care effect, like by human consumer deeply.Market milk acid bacteria drink is mainly divided into viable type and the large class of non-viable type two.Viable type is formed by fermentation, containing a large amount of viable bacteria, proves to produce useful physiological action to human body through science; But not viable type does not almost have health-care effect.So the exploitation of viable bacteria beverage certainly will become the study hotspot of lactobacillus drink.
At present, in the lactobacillus drink developed both at home and abroad, what the fermented bacterium use used was more is lactobacterium casei (Lactobacillus casei), Lactobacterium acidophilum (Lactobacillus acidophilus), plant lactobacillus (Lactobacillus plantarum), lactobacillus paraceasi (Lactobacillus paracasei) etc., minority is had also to adopt bifidus bacillus, as animal bifidobacteria (Bifidobacterium animal) etc., yet there are no patent and the bibliographical information of bifidus longum bb active fermentation beverage.And bifidus bacillus (Bifodobacterium spp.) is the Exemplary beneficial milk-acid bacteria colonized in humans and animals enteron aisle, it can play biological barrier, nutrition, immunity to host, delays senility, the physiological action such as antitumor.Produce viable bacteria beverage with bifidobacterium fermentation and greatly can strengthen its nourishing function, improve added value of product.
In the lactobacillus drink developed, fermentation raw material used mainly contains: 1) Dairy products, as cow's milk, soya-bean milk, coconut palm breast etc.; 2) fruit and vegetable product class, as Radix Dauci Sativae, apple, corn, pumpkin, red date, tomato, small berries etc.; 3) nuts, as peanut, walnut etc.Yet there are no with the bifidus bacillus drink listing that is fermentation raw material of sea-buckthorn matrimony vine compound juice.
Sea-buckthorn formal name used at school (Hippophae rhamnoides Linn), having another name called vinegar willow, Mo Ci, is the mature fruit of Elaeangnaceae hippophae plant sea-buckthorn, and be medicinal and edible plant, fruit contains abundant nutritive substance and biologically active substance.Especially the vitamins C of rich content in fruit, has promoter action to the propagation of bifidus bacillus.
Matrimony vine (Gycium barbarum), as the traditional rare traditional Chinese medicine of China and Important Economic crop, has multiple physiologically active and nourishing function.Clinical medicine shows that matrimony vine has significant curative effect to diabetes, hypertension, central retinitis, optic atrophy, ephritis and hepatitis etc., and the lycium barbarum polysaccharide in matrimony vine has growth promoting function to bifidus bacillus.
Make viable bacteria beverage with bifidobacterium fermentation sea-buckthorn matrimony vine compound juice, both can play sea-buckthorn, the nutrition of matrimony vine and physiological function, the health-care effect of bifidus bacillus can have been strengthened again, the viable bacteria drink of high added value will be become.
Summary of the invention
The object of this invention is to provide a strain have the bifidus longum bb of excellent probiotic properties and preparing the application in living bifidobacteria fermented drink.
Bifidus longum bb (Bifidobacterium longum) BF-10 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 06 21st, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .7789.
Bifidus longum bb CGMCC № .7789 provided by the invention, has tolerance digestive tube inverse ring border, biological antagonist and the health-care effect such as anti-oxidant, can be used for preparing bifidobacteria viable bacteria beverage.
The preparation method that the present invention also provides a kind of bifidus longum bb to ferment sea-buckthorn matrimony vine compound juice viable bacteria beverage, be seeded in the composite fruit juice substratum containing sea-buckthorn wolfberry juice compound juice, milk powder and sucrose solution after bifidus longum bb (Bifidobacterium longum) BF-10 is activated, at a certain temperature after Anaerobic culturel certain hour, terminate fermentation, obtain bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage.
In described method, sea-buckthorn matrimony vine compound juice fermention medium is prepared as follows: get the water mixing making beating that fresh sea-buckthorn and mass ratio are 1: 1, by four layers of filtered through gauze, obtain the juice of Fructus Hippophae that mass percent is 50%; Precise wolfberry fruit, adds in triangular flask by material quality than 1: 10, and in 85 DEG C of water-baths, soak making beating after two hours, four layers of filtered through gauze, obtain the wolfberry fruit extract that effective constituent mass percent is 10%; Above-mentioned juice of Fructus Hippophae and wolfberry fruit extract to be mixed by volume at 2: 1, obtains fruit juice blends; Sucrose 75g is dissolved in 100mL water and obtains sucrose solution; In 1L water, add 10g skimmed milk powder, abundant stirring and dissolving, obtain degreasing reconstituted milk; According to fruit juice blends 50%, sucrose solution 12%, degreasing reconstituted milk 10%, water 28% prepares composite fruit juice substratum; Under 8MPa condition, carry out first time homogeneous with clarifixator, under 12MPa condition, carry out second time homogeneous, and with sodium bicarbonate (N ahCO 3) regulate about 6.0 of its pH; Finally, in composite fruit juice fermention medium, fill nitrogen, make Redox potential be down to below-0.03eV, and at 115 DEG C, high pressure steam sterilization 10min.
In described method, the leavening temperature of bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage is 38-42 DEG C, and fermentation time is 24-26h, and initial ph value is 6.0, and inoculum size is 1 × 10 6cfu/mL.
The present invention utilizes bifidus longum bb BF-10CGMCC № .7789 as fermentation strain, have selected nutritious and containing can promote that the juice of Fructus Hippophae of the physiologically active substance that bifidus bacillus breeds and wolfberry fruit extract are fermentation raw material simultaneously, and add a certain amount of sucrose and skimming milk, prepare a kind of bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage.Composite fruit juice is after the fermentation of this bifidobacterium strains, and viable count can reach 8.7 × 10 10cFU/mL, also can produce acetic acid, lactic acid simultaneously, the multiple meta-bolites with important physiologically active such as vitamin B group, the sense organ making product formation good and nutritional quality.Finished product, through sterile filling, can preserve 19 days under 4 DEG C of conditions, can preserve 8 days under room temperature (22 DEG C).Bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage color and luster prepared by the present invention is gentle, mouthfeel is good, local flavor is good, viable count is high, shelf-lives is longer, both can play sea-buckthorn, the nutrition of matrimony vine and physiological function, can strengthen again the health-care effect of bifidus bacillus, be the health promoting beverage with more physiological function.
Accompanying drawing explanation
Fig. 1 is bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria drink sample (one of packaged form) of the present invention.
Fig. 2 is under different reserve temperature, the number of active bifid bacteria situation map over time in fermented drink.
Embodiment
Experimental technique described in following embodiment, if no special instructions, is ordinary method.
The isolation identification of embodiment 1, bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789
1, the screening of excellent bifidobacterium strains
For obtaining the excellent bifidobacterium strains in inverse ring border, digestion resistant road, to derive from 5 strain bifidus bacilluss of healthy breast milk infant faeces and long lived elder enteron aisle (La Yisu village, hotan Yutian County) for starting strain, adopt MRS liquid culture medium, simulation human gastrointestinal tract environment, studies against environmental characteristics its digestion resistant road.Starting strain numbering and source are in table 1.
(1) sour tolerance test: based on the PBS damping fluid of pH7.0, pH3.0 is adjusted to again with the hydrochloric acid of 37%, activated the bifidus bacillus BF-10 liquid culture in 3 generations by the inoculum size access of 5% after 121 DEG C × 20min sterilizing, 37 DEG C of Anaerobic culturel, respectively at 0 and 2h sampling, roll tube method with Heng Gaite anaerobism and measure viable count, and calculate survival rate.
(2) bile salt tolerance test: it is in the MRS liquid culture medium of 0.5% that the liquid culture after actication of culture 3 generation is contained Glycocholate sodium by 2% inoculum size access, sampling and measuring viable count after 37 DEG C of Anaerobic culturel 12h; Simultaneously not contain the MRS liquid culture medium of bile salt in contrast, measure viable count.Calculate survival rate.
(in the 1000mL) composed as follows of modified MRS culture medium used: glucose 20g, beef extract powder 10g, peptone 10g, yeast leaching powder 5g, corn cream 3g, anhydrous sodium acetate 5g, triammonium citrate 2g, dipotassium hydrogen phosphate 2g, manganous sulfate 0.25g, magnesium sulfate 0.58g, Cys hydrochloric acid 0.4g, tween-80 1mL.
(3) resistance to stomach en-test: based on the phosphoric acid buffer of pH7.0, with the hydrochloric acid of volume fraction 37%, it is adjusted to pH3.0 respectively, add 0.5% stomach en-again as simulated gastric fluid, by the liquid culture after 5% inoculum size access actication of culture 3 generation, calculate survival rate respectively at 0 and 2h sampling and measuring viable count.
(4) resistance to trypsin test: based on 1/15mol/mL, pH7.0 phosphoric acid buffer, adds 10mg/mL trypsinase as simulated intestinal fluid, by the bacterium amount that the connects aseptic access bacterium liquid of 5%, act under 37 DEG C of anaerobic conditions, respectively at 0h and 12h sampling and measuring viable count, calculate survival rate.
The tolerance of table 1 five strain strain subject to digestive tube inverse ring border compares
As can be seen from table 1 the selection result, bacterial strain BF-10 survival rate after acid treatment is 96.1%, after bile salt and pepsin, survival rate is all more than 86%, after trypsin treatment 12h, bacterial strain increases to some extent, illustrate that bifidus bacillus BF-10 all has very strong resistibility to acid, bile salt, stomach en-and trypsinase, and the survival rate after four process is apparently higher than other test strains.Can find out that bacterial strain BF-10 has the great potential as probiotics preparation by result, therefore to choose bifidus bacillus BF-10 be target strain excellent.
2, the qualification of strain excellent
Morphological observation, API 20A biochemical reactions and 16S rDNA sequential analysis is adopted to carry out taxonomy qualification to aimed strain BF-10.Bacterial strain BF-10 is well-grown in improvement solid MRS anaerobism pipe, and bacterium colony is in white, and circular, edge is neatly more smooth, without obvious bacterium colony under aerobic conditions; By gramstaining after picking colony dilution, basis of microscopic observation finds: BF-10 somatic cells form is G +, present shaft-like, part, in the bifurcation structure such as V or Y-shaped, without gemma, is not moved, irregular alignment.According to API Bacteria Identification standard, API 20A anaerobism identification systems are used to carry out 20 kinds of sugar alcohol fermentation tests to bacterial strain BF-10.To table look-up analytical reagent bar reaction result, tentatively determine that BF-10 belongs to genus bifidobacterium (Bifodobacterium spp.).Extract the DNA genome of bacterial strain BF-10, obtain the DNA sequencing fragment (see sequence table) of 1349bp through pcr amplification, and reclaimed purifying, transfer to Beijing six directions Hua Da Gene Tech. Company Limited to check order.Sequence analysis is carried out by the Genbank of sequencing result on NCBI, use Mega3.1 software building phylogenetic tree, find that bacterial strain BF-10 and Bifidobacterium longum subsp.infantis is in same subbranch, sibship is nearest, and similarity is up to 99.2%; On this basis, then combining form observe and API 20A biochemical identification result, determine that bacterial strain BF-10 is bifidus longum bb (Bifidobacterium longum).
Bifidus longum bb (Bifidobacterium longum) BF-10 has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center and (has been called for short CGMCC on 06 21st, 2013, address is: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica), preservation registration number is: CGMCC № .7789.
The efficacy assessments of embodiment 2, bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789
1, biological antagonist merit rating
Adopt the double-deck agar diffusion method of Oxford cup, detect the fungistatic effect of bacterial strain BF-10 fermented supernatant fluid.By mix-incubation experiment, detect the biological antagonist ability of bacterial strain BF-10 somatic cells.
(1) the fungistatic effect analysis of strain fermentation supernatant liquor: bifidus longum bb BF-10 37 DEG C of Anaerobic culturel 24h, by fermented liquid with the centrifugal 15min of 5000rpm/min, collect supernatant liquor, be divided into two groups, one group as former fermented supernatant fluid sample, one group is neutralized to pH about 7.0 through 1M NaOH, as in and secondary fermentation supernatant samples.Indicator final concentration is 10 7cfu/mL, after adding 100 μ L samples, cultivates in indicator optimum condition, measure antibacterial circle diameter (mm) in the cup of Oxford.
Table 2 result shows, former fermented supernatant fluid all shows fungistatic effect to a certain degree to the tested indicator of 8 bacterium, wherein comparatively strong to the antagonistic action of intestinal bacteria 1.90, streptococcus aureus 1.128, streptococcus aureus 1.169, Listeria monocytogenes 35152 (4b), Bacillus coagulans, antibacterial circle diameter is all at more than 20mm.Fermented supernatant fluid is after neutralizing acid process, only can still keep certain fungistatic effect to streptococcus aureus 1.128 and Listeria monocytogenes 35152, this illustrates bacterial strain BF-10 except except exocytosis organic acid, also metabolism can producing some other antibacterial substances, as bacteriocin etc.Illustrate further, the biological antagonist ability of bacterial strain BF-10 is excellent.
The bacteriostatic action of table 2 bacterial strain BF-10 fermented supernatant fluid
(2) the Ant agonism analysis of somatic cells: inoculate tested indicator (totally 8 kinds respectively by 1% inoculum size in MRS liquid culture medium, in table 2) and bifidus longum bb BF-10, mixing, sample after being placed in 37 DEG C of Anaerobic culturel 24h, adopt the corresponding selective medium of each indicator, by colony counting method, live bacterial count is carried out to each indicator, investigate the survival condition of each indicator.And with the pure growth of independent tested indicator in MRS liquid nutrient medium as contrast, culture condition is identical.Wherein, streptococcus aureus selective medium used is Baird-Parker substratum, and listeria bacteria selective medium used is listeria spp color developing culture medium, and intestinal bacteria selective medium used is Yihong methylene blue substratum.Result shows, each indicator control group presents and well grows situation in MRS liquid nutrient medium, and after cultivating 24h, viable count logarithmic value is all more than 8.After bifidus bacillus BF-10 mixed culture, the growth of each indicator is all subject to obvious suppression.Wherein, the viable count of intestinal bacteria 1.90, Bacillus coagulans, streptococcus aureus 1.169 and 1.128 declines comparatively obviously (2.1-2.5log).These results suggest that bifidus longum bb BF-10 all has obvious antagonistic action to tested 8 strain bacterium, this may be relevant with the nutrient competition between them or competition for space, and BF-10 is as relevant in bacteriocin with other antibacterial substances at process of growth generation organic acid.
In sum, the somatic cells of bacterial strain BF-10 and fermented supernatant fluid bacterium show very strong biological antagonist ability, use it for and prepare viable bacteria fermentation beverage, largely can improve the security of product.
2, anti-oxidation efficacy evaluation
Removed by ultra-oxygen anion free radical, DPPH (1,1-phenylbenzene-2-trinitrophenyl-hydrazine) free radical scavenging, 4 tests such as Hydroxyl radical-scavenging and reducing power mensuration, study the resistance of oxidation of the somatic cells of bacterial strain BF-10, thalline cell-free extract and cell free fermentation supernatant liquor.
(1) the producing of sample: mainly comprise 3 class samples, be respectively somatic cells, thalline cell-free extract and cell free fermentation supernatant liquor.By bifidus longum bb BF-10 at 37 DEG C of Anaerobic culturel 24h, get fermented liquid with the centrifugal 15min of 5000rpm/min, collect supernatant liquor, be cell free fermentation supernatant samples, collect somatic cells simultaneously, and wash 3 times with distilled water, finally with distilled water, its bacterium number is adjusted to 10 9cells/mL, gained bacteria suspension is divided into two groups, one group as somatic cells sample, one group is put in sonicated cells in ice bath, broken liquid in the centrifugal 10min of 6000r/min, collect supernatant, be thalline cell-free extract sample.
(2) superoxide anion test is removed: adopt assay NBT photoreduction to measure its resistance of oxidation.Get 4.5mL, 0.1mol/L Tris-HCl damping fluid (pH8.2), in test tube, adds lmL successively, 1.0mmol/L ethylenediamine tetraacetic acid (EDTA) (hot water dissolving), 1.0 samples, 2.4mL distilled water, adds 2mi pyrogallol after 25 DEG C of reaction 10min.After 25 DEG C of reaction 60min, add 100 μ L12mol/L HCl termination reactions.Absorbancy (As) is measured at wavelength 325nm place.Blank tube replaces sample with 1.0mL distilled water, and the same sample hose of working method, records absorbancy (Ac).All replicate(determination) is averaged for 3 times above, and 3 times mensuration is averaged.Wherein, the preparation of the Tris-HCl of 0.1mol/L, pH 8.2: 0.346gTris alkali, constant volume to 1000ml, with concentrated hydrochloric acid intermodulation to pH8.2.Clearance rate is by following formulae discovery: clearance rate/%=[(Ac-As)/Ac] × 100
(3) test of DPPH free radical is removed: get 4.0mL0.1mmol/LDPPH solution in test tube, add 2.0mL sample and 1.0mL50% ethanol, in the dark the centrifugal 20min of 12000r/min after reaction 60min, gets supernatant liquor and surveys absorbancy (Aj) at wavelength 517nm place.Blank tube replaces sample with 2.0mL distilled water, and the same sample hose of working method, records absorbancy (Ai).All replicate(determination) is averaged for 3 times above, and 3 times mensuration is averaged.Clearance rate is by following formulae discovery: clearance rate/%=[(Ai-Aj)/Ai] × 100
(4) scavenging hydroxyl test: the phosphoric acid buffer of the 0.05mol/L adding 1mL in test tube respectively, pH7.4, the phenanthroline of 0.5mL, 6mmol/L, fully after mixing, add 0.5mL, the FeSO4 solution of 6mmol/L, mixes after adding immediately.Then add the sample solution of 0.5mL wherein, mixing, then add 0.5mL, the H of 0.1% 2o 2, finally with distilled water, volume is added to 4mL, does blank assay simultaneously.Another doing again is damaged pipe and is not damaged pipe, wherein, adds 0.5mL, the H of 0.1% in damage pipe 2o 2, do not add sample.Do not damage pipe and do not add H 2o 2, also do not add sample.Finally all volume is added to 4mL.In 37 DEG C of insulation 1h, survey its absorbancy at 536nm wavelength place.Clearance rate is by following formulae discovery: clearance rate/%=[(Ai-A)/(A 0-A)] × 100, A be the absorbancy of solution when not adding sample; A 0for not application of sample and H 2o 2time solution absorbancy; Ai is the absorbancy of solution when adding sample.
(5) reducing power measures: get 0.5mL sample and add concentration 0.2mol/L, the phosphate buffer solution 0.5mL of pH6.6 and 1g/100mL Tripotassium iron hexacyanide 0.5mL, in 50 DEG C of water-bath 20min, cools rapidly.Add 10g/100mL trichoroacetic acid(TCA) 0.5mL again, the centrifugal 5min of 3000rpm/min, gets supernatant liquor 1mL, adds 1mL distilled water and 1mL, 0.1g/100mL iron trichloride, mixes.After 10min, under 700nm, measure its absorbancy.The reducing power of absorbancy larger expression testing sample is stronger.
The anti-oxidant activity analysis of table 3 bacterial strain BF-10
Table 3 result shows, somatic cells up to 94.53%, illustrates that the active substance of scavenging hydroxyl clearance rate is mainly present in the somatic cells surface of bifidus longum bb BF-10 to Scavenging action to hydroxyl free radical; Thalline cell-free extract, to superoxide anion clearance rate higher (97.67%), illustrates the intracellular organic matter of the active substance mainly baby's somatic cells removing ultra-oxygen anion free radical; Cell free fermentation supernatant liquor is the highest to DPPH clearance rate, can reach nearly 93%; This illustrates that the active substance removing DPPH free radical may be metabolic secretion thing.In addition, the reducing power of cell free fermentation supernatant liquor is the strongest, and the active substance that there is reducing power outside somatic cells born of the same parents is described.This result of study shows, and the somatic cells of bacterial strain BF-10 and mucilage secretion all have stronger resistance of oxidation, in further product development, can consider to produce viable bacteria beverage using this bacterial strain as starter bacterial strain, play its anti-oxidation efficacy comprehensively.
In sum, bifidus longum bb BF-10 is except having very strong tolerance to digestive tube inverse ring border, also can play Ant agonism to multiple harmful microorganism, and somatic cells and mucilage secretion all have very strong oxidation-resistance, physiological function is obvious, has the potentiality as probiotic strain application and exploitation series product.
The preparation method of embodiment 3, bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789 fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage
The present invention utilizes embodiment 1 and 2 gained bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789 to ferment sea-buckthorn matrimony vine compound juice to prepare bifidobacteria viable bacteria beverage, and preparation method is as described below:
1, bifidus bacillus seed is prepared
(1) preparation of generation seed: bifidus longum bb (Bifidobacterium longum) BF-10 is inoculated in MRS liquid culture medium, at 37 DEG C of Anaerobic culturel 24h-48h, obtains generation seed.
(2) two generation seed preparation: (late log phase is that logarithmic phase closes to an end and turnover time period coming to the ripening period generation seed to be seeded in MRS liquid culture medium 37 DEG C of Anaerobic culturel 14-16h (to late log phase).By measuring the growth curve of practical condition, judge the time range of late log phase), obtain s-generation seed.
(3) preparation of three generations's seed: by two generation seed be seeded in Anaerobic culturel 14h (to late log phase) in modified MRS culture medium and obtain third generation seed.Third generation seed uses as cultivation seed.
2, the preparation of sea-buckthorn matrimony vine compound juice fermention medium
Get the tap water mixing making beating that fresh sea-buckthorn and mass ratio are 1: 1, by four layers of filtered through gauze, obtain the juice of Fructus Hippophae that mass percent is 50%; Precise wolfberry fruit, adds in triangular flask by material quality than 1: 10, and in 85 DEG C of water-baths, soak making beating after two hours, four layers of filtered through gauze, obtain the wolfberry fruit extract that effective constituent mass percent is 10%; Above-mentioned juice of Fructus Hippophae and wolfberry fruit extract to be mixed by volume at 2: 1, and with sodium bicarbonate (N ahCO 3) regulate about 6.0 of its pH, obtain fruit juice blends; Sucrose 75g is dissolved in 100mL water and obtains sucrose solution; In 1L water, add 10g skimmed milk powder, abundant stirring and dissolving, obtain degreasing reconstituted milk.According to fruit juice blends 50%, sucrose solution 12%, degreasing reconstituted milk 10%, water 28% is prepared, and under 8MPa condition, carries out first time homogeneous with clarifixator, carries out second time homogeneous under 12MPa condition, obtains composite fruit juice fermention medium; Finally, in composite fruit juice fermention medium, fill nitrogen, make Redox potential be down to below-0.03eV, and at 115 DEG C, high pressure steam sterilization 10min.
3, bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage is prepared
Seed step 1 obtained is with 2% inoculum size (1 × 10 6cfu/mL) be seeded to containing in sea-buckthorn matrimony vine composite fruit juice substratum, 38-42 DEG C of Anaerobic culturel is about 24-26h, is 4.2-4.5 to pH.Fermentation ends, obtains sea-buckthorn matrimony vine bifidobacterium fermentation beverage.By fermented drink packing, sterile filling as shown in Figure 1, in 4 DEG C of storages.Heng Gaite anaerobism rolls tube method counting viable count can reach 8.7 × 10 10cFU/mL.
The quality of embodiment 4, bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789 fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage and shelf-lives evaluation
1, quality evaluation
Evaluate based on physical and chemical index mensuration and the quality of sensory evaluation to prepared living bifidobacteria fermented drink.
(1) physical and chemical index measures: carry out following index determining, 1 to bifidus bacillus sea-buckthorn matrimony vine compound juice fermentation activity beverage prepared by embodiment 3) protein content: with reference to GB 50095-2010 " mensuration of Protein in Food "; 2) soluble solid content: with reference to GB 12143.1-88 the measuring method of soluble solid " in the soft drink "; 3) reducing sugar content: with reference to GB/T 5009.7-2003 the mensuration of reducing sugar " in the food "; 4) total sugar content: Phenol sulfuric acid procedure; 5) total acid content: with reference to GB/T 12456-2008 the measuring method of total acid " in the food " (in lactic acid), 6) pH value, measured by pH value electrode.The results are shown in Table 5, can find out, the physical and chemical index of viable bacteria beverage prepared by embodiment 3 all meets the requirement of standard GB/T 16321-2003 " lactobacillus drink hygienic standard ".
The physical and chemical index analysis of viable bacteria beverage prepared by table 5 embodiment 3
(2) sensory evaluation: carry out organoleptic analysis to bifidus bacillus sea-buckthorn matrimony vine compound juice fermentation activity beverage prepared by embodiment 3, adopts fuzzy mathematics seven degree of scaling laws, forms scoring group by 10 people through training.Comment domain is V=(1,2,3,4,5,6,7), 1: the most difficult acceptance; 2: more difficult acceptance; 3: slightly difficult acceptance; 4: accept reluctantly; 5: more easily accept; 6: acceptant; 7: the most easily accept.The evaluation field U=(color and luster, fragrance, acidity, sugariness, bitter taste, structural state) of fermented juice, the weighted value of its correspondence is X=(0.15,0.20,0.20,0.15,0.20,0.10).The results are shown in Table 6, can find out this beverage have sea-buckthorn and Chinese wolfberry fruit aroma and bifidobacterium fermentation after distinctive fragrance, its color and luster, structural state, sugariness score are all more than 6.0.Its integrated sensory must be divided into 6.28, is a kind of drink easily accepted by the public.
The sensory evaluation Score Lists of viable bacteria beverage prepared by table 6 embodiment 3
2, shelf-lives evaluation
Viable bacteria beverage is placed in respectively 4 DEG C and room temperature (22 DEG C) preservation, every sampling in 1 day, Heng Gaite anaerobism rolled tube method counting bifidus bacillus.With reference to GB 16321-2003 " lactobacillus drink hygienic standard " to the minimum limit standard of viable count of lactobacillus in lactobacillus drink, with viable count lower than 1.0 × 10 6cFU/mL judges shelf-lives terminal.Result as shown in Figure 2, can be found out, the number of active bifid bacteria amount in 4 DEG C of storages of viable bacteria beverage prepared by embodiment 3 declines slowly, can ensure that in storage 19 days, the number of active bifid bacteria is 1.0 × 10 6more than CFU/mL; And the number of active bifid bacteria amount viable count declines rapidly in room temperature storage process, only 8 days can be preserved.More than show, the shelf-lives of reserve temperature on viable bacteria drink has remarkably influenced, and as a kind of viable bacteria drink shorter without the shelf-lives of re-sterilise, this fermented drink is preferably takes cold chain sales mode.

Claims (7)

1. bifidus longum bb (Bifidobacterium longum) BF-10CGMCC № .7789.
2. producing a method for bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage, is fermentation bifidus longum bb according to claim 1 (Bifidobacterium longum) BF-10CGMCC № .7789, the viable bacteria beverage obtained.
3. method according to claim 2, is characterized in that: described fermention medium is prepared as follows: get the water mixing making beating that fresh sea-buckthorn and mass ratio are 1: 1, by four layers of filtered through gauze, obtain the juice of Fructus Hippophae that mass percent is 50%; Precise wolfberry fruit, adds in triangular flask by material quality than 1: 10, and in 85 DEG C of water-baths, soak making beating after two hours, four layers of filtered through gauze, obtain the wolfberry fruit extract that effective constituent mass percent is 10%; Juice of Fructus Hippophae is mixed according to a certain volume with wolfberry fruit extract, obtains fruit juice blends; Sucrose 75g is dissolved in 100mL water and obtains sucrose solution; In 1L water, add 10g skimmed milk powder, abundant stirring and dissolving, obtain degreasing reconstituted milk; By fruit juice blends, sucrose solution, degreasing reconstituted milk and water mix by a certain percentage; Under 8MPa condition, carry out first time homogeneous with clarifixator, under 12MPa condition, carry out second time homogeneous, and with sodium bicarbonate (N- ahCO 3) regulate about 6.0 of its pH, obtain composite fruit juice fermention medium; Finally, in composite fruit juice fermention medium, fill nitrogen, make Redox potential be down to below-0.03eV, and at 115 DEG C, high pressure steam sterilization 10min.
4. method according to claim 3, is characterized in that: fermention medium consists of fruit juice blends 50%, sucrose solution 12%, degreasing reconstituted milk 10%, water 28%; Described percentage composition is percent by volume.
5. method according to claim 3 or 4, is characterized in that: in described fruit juice blends, the composite ratio of juice of Fructus Hippophae and wolfberry fruit extract is 2: 1.
6. method according to Claims 2 or 3 or 4 or 5, is characterized in that: described leavening temperature is 38-42 DEG C, and fermentation time is 24-26h, and initial ph value is 6.0, and inoculum size is 1 × 10 6cfu/mL.
7. bifidobacterium fermentation sea-buckthorn matrimony vine compound juice viable bacteria beverage prepared by the method in claim 2-6 described in any one.
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104940450A (en) * 2015-05-22 2015-09-30 北京联华大通教育科技有限公司 Health beverage for enhancing body immunity and preparation method thereof
CN106947726A (en) * 2017-05-25 2017-07-14 华中农业大学 The high density fermentation and method for cold air drying of a kind of Lactobacillus casei
CN110452829A (en) * 2019-03-24 2019-11-15 广西大学 Bifidobacterium longum bacterial strain and its application
CN111557378A (en) * 2020-05-20 2020-08-21 江南大学 Method for preparing fermented feed by using bifidobacterium longum
TWI742406B (en) * 2019-07-19 2021-10-11 大陸商內蒙古伊利實業集團股份有限公司 Anti-oxidation food composition and pharmaceutical composition with strain of lactic acid bacterium
CN113817622A (en) * 2020-06-18 2021-12-21 青岛蔚蓝生物股份有限公司 Bifidobacterium longum with effect of relieving human cytogene toxicity of carcinogen and application thereof
TWI752334B (en) * 2019-07-19 2022-01-11 大陸商內蒙古伊利實業集團股份有限公司 Food composition and pharmaceutical composition with anti-oxidation fermentation metabolites of lactic acid bacterium

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206599A (en) * 2011-04-11 2011-10-05 天津科技大学 Oxygen-resistant acid-resistant Bifidobacterium longum
CN104138527A (en) * 2014-08-13 2014-11-12 胡广 Biological fermentation composition with anti-cancer effect and application of biological fermentation composition

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102206599A (en) * 2011-04-11 2011-10-05 天津科技大学 Oxygen-resistant acid-resistant Bifidobacterium longum
CN104138527A (en) * 2014-08-13 2014-11-12 胡广 Biological fermentation composition with anti-cancer effect and application of biological fermentation composition

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JING WANG ET AL.: "In vitro fermentation of xylooligosaccharides from wheat bran insoluble dietary fiber by Bifidobacteria.", 《CARBOHYDRATE POLYMERS》 *
徐文生等: "环境因素对长双歧杆菌CICC6069生物膜生成的影响", 《中国食品学报》 *
黄巧芬: "优良双歧杆菌的筛选及其生理功能研究", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 *

Cited By (9)

* Cited by examiner, † Cited by third party
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CN104940450A (en) * 2015-05-22 2015-09-30 北京联华大通教育科技有限公司 Health beverage for enhancing body immunity and preparation method thereof
CN106947726A (en) * 2017-05-25 2017-07-14 华中农业大学 The high density fermentation and method for cold air drying of a kind of Lactobacillus casei
CN110452829A (en) * 2019-03-24 2019-11-15 广西大学 Bifidobacterium longum bacterial strain and its application
TWI742406B (en) * 2019-07-19 2021-10-11 大陸商內蒙古伊利實業集團股份有限公司 Anti-oxidation food composition and pharmaceutical composition with strain of lactic acid bacterium
TWI752334B (en) * 2019-07-19 2022-01-11 大陸商內蒙古伊利實業集團股份有限公司 Food composition and pharmaceutical composition with anti-oxidation fermentation metabolites of lactic acid bacterium
CN111557378A (en) * 2020-05-20 2020-08-21 江南大学 Method for preparing fermented feed by using bifidobacterium longum
CN111557378B (en) * 2020-05-20 2022-09-27 江南大学 Method for preparing fermented feed by using bifidobacterium longum
CN113817622A (en) * 2020-06-18 2021-12-21 青岛蔚蓝生物股份有限公司 Bifidobacterium longum with effect of relieving human cytogene toxicity of carcinogen and application thereof
CN113817622B (en) * 2020-06-18 2023-05-26 青岛蔚蓝生物股份有限公司 Bifidobacterium longum with effect of reducing genotoxicity of carcinogen to human cells and application thereof

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