CN102329763A - Lactobacillus helveticus H9 and application of Lactobacillus helveticus H9 to preparation of fermented milk - Google Patents
Lactobacillus helveticus H9 and application of Lactobacillus helveticus H9 to preparation of fermented milk Download PDFInfo
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Abstract
The invention relates to application of Lactobacillus helveticus H9 to fermented milk products. A Lactobacillus helveticus H9 fermented milk product is prepared by inoculating a strain containing the Lactobacillus helveticus H9 into fresh milk (cow milk, horse milk and soybean milk) or reconstitued milk by an appropriate process. The fermented milk has angiotensin-converting enzyme inhibitory activity (60 to 75 percent) and is rich in bioactive peptides, namely Val-Pro-Pro (2.8 to 3.2mu M) and Ile-Pro-Pro (2.2 to 2.7mu M).
Description
[technical field]
The invention belongs to the microbial technique Application Areas.More specifically, the present invention relates to a kind of lactobacterium helveticus (Lactobacillus helveticus) H9, also relate to the purposes of said lactobacterium helveticus H9 in the preparation fermented-milk.
[background technology]
Though milk-acid bacteria self possesses certain amino acid synthesis capability; But still need through proteolyze system degraded exogenous protein or peptide; Satisfy the nutritional needs in the process of growth; Be accompanied by the hydrolysate that will contain biologically active peptides (hypotensive, reducing blood-fat, anti-oxidant, bacteriostatic peptide, raising immunity etc.) and be discharged into outside the born of the same parents, this characteristic is also most important with the functional performance and the local flavor of cultured milk prod.
The lactic bacterial protein hydrolysis system also there are differences between the bacterial classification even between the bacterial strain.In the milk-acid bacteria family, lactobacterium helveticus (Lactobacillus helveticus) has stronger milk-protein hydrolytic activity, also is the maximum milk-acid bacteria of antihypertensive active research.
Lactobacterium helveticus H9 relies on 259 strain lactobacterium helveticus in Agricultural University of the Inner Mongol's milk-acid bacteria resources bank to be the basis; Suppressing activity, Val-Pro-Pro content, Ile-Pro-Pro content and fermentation time with the ACE of fermented-milk is that the 1 strain bacterium source that filters out of leading indicator is clear and definite, and genetic background clearly has high ACE and suppresses active probiotic lactobacillus.
Zhao Wen waits the people quietly in " selection of internal control gene in the real-time fluorescence quantitative PCR "; " microbiology circular "; 37 (12), having described with lactobacterium helveticus H9 among the P1825-1829 (2010) is research object, uses the real-time fluorescence quantitative PCR technology; Estimated 5 kinds of stability that internal control gene commonly used is expressed, thereby obtained the most stable internal control gene into real-time fluorescence quantitative PCR analysis use.
At present, also there are not bibliographical information lactobacterium helveticus H9 biological characteristics, cultural method and its application in the preparation milk-product.Therefore, the inventor has accomplished the present invention finally through lot of experiments.
[summary of the invention]
[technical problem that will solve]
The purpose of this invention is to provide a kind of lactobacterium helveticus (Lactobacillus helveticus) H9.
Another object of the present invention provides and a kind ofly contains the purposes in the fermented-milk of lactobacterium helveticus H9 in preparation.
[technical scheme]
The present invention realizes through following technical proposals.
The present invention relates to a kind of lactobacterium helveticus (Lactobacillus helveticus) H9; This bacteria strain on April 28th, 2011 in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms (CGMCC) preservation, preserving number is CGMCC4811.
The invention still further relates to the purposes of described lactobacterium helveticus H9 in the preparation fermented-milk.
A preferred embodiment of the invention, the preparation process of fermented-milk that contains lactobacterium helveticus H9 is following:
In 920-960 weight part sweet milk or recombined milk, add 40-80 weight part sugar; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to temperature 60-65 ℃, under homogenization pressure 15-25MPa, carries out the homogeneous liquid that the obtains 5-15s that under temperature 120-140 ℃, sterilizes again then homogeneous 10-30 minute; Be cooled to temperature 37-42 ℃ then, inoculation 0.02-0.10 weight part 1 * 10
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs, then at temperature 37-42 ℃ of bottom fermentation 7-8h, with inorganic base aqueous solution with the pH regulator of its fermented liquid to 4.2-4.6, at last at temperature 4-8 ℃ of following after-ripening 8-12h, obtain described lactobacterium helveticus H9 fermented-milk.
According to another kind of preferred implementation of the present invention, described sweet milk is selected from cow's milk, horse breast or soya-bean milk.
According to another kind of preferred implementation of the present invention, described recombined milk is with 120-150 weight part milk powder and the preparation of 850-880 weight part softening water.
According to another kind of preferred implementation of the present invention, described sugar is selected from sucrose, glucose, fructose, semi-lactosi, lactose, SANMALT-S, starch, dextrin or glycogen marsh-mallow.
According to another kind of preferred implementation of the present invention, described mineral alkali is selected from Pottasium Hydroxide, sodium hydroxide or yellow soda ash.
According to another kind of preferred implementation of the present invention, in sweet milk or recombined milk, add the bacterial classification of delivering directly in said newborn gross weight 0.01-1.0% thermophilus streptococcus and lactobicillus bulgaricus.
According to another kind of preferred implementation of the present invention, described fermented-milk contains 2 * 10
7Cfu/g-1 * 10
8Cfu/g lactobacterium helveticus H9, the content of VPP are 3.0-3.3 μ M, and the content of IPP is 2.2-2.5 μ M.
According to another kind of preferred implementation of the present invention, in the after-ripening step of fermented-milk or in the fermented-milk that is obtaining, add in said fermented-milk gross weight 0.5-1.0% thickening material or essence.
Below the present invention will be described in more detail.
The present invention relates to a kind of lactobacterium helveticus H9, this bacteria strain on April 28th, 2011 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC4811.
The biological characteristics of described lactobacterium helveticus H9 is following:
The invention still further relates to the purposes of described lactobacterium helveticus H9 in the preparation fermented-milk.
A preferred embodiment of the invention, the preparation process of fermented-milk that contains lactobacterium helveticus H9 is following:
In 920-960 weight part sweet milk or recombined milk, add 40-80 weight part sugar, remove impurity at sugar dissolving after-filtration, then it is contained sugared emulsion is heated to temperature 60-65 ℃.
Described sweet milk is selected from cow's milk, horse breast or soya-bean milk.
Milk property is flat, and it is sweet to distinguish the flavor of.Tonifying deficiency, beneficial lung stomach, the ease constipation of promoting the production of body fluid.Suitable physique is thin and weak, insufficiency of vital energy and blood, and people malnutritive and body void after being ill is edible; Suitable constipation of old people person, upgrowth and development of children phase, diabetic subject, Patients with Sjogren Syndrome, essential hypertension, coronary heart disease, arteriosclerosis, hyperlipidaemia person eat.
The horse milkiness is cool, and is noisy sweet.Enriching blood and moistening for dryness relieves heat and thirst.The horse breast contains the N,O-Diacetylmuramidase of high level, reaches 0.3-1.0mg/ml.N,O-Diacetylmuramidase has germicidal action to gram-positive bacteria, the β-1 between ability hydrolyzing N-acetylmurami and kharophen deoxyglucose, and the 4-glycosidic link makes cell wall rupture, causes bacterial death.N,O-Diacetylmuramidase and microbiotic have share synergy.N,O-Diacetylmuramidase has antivirus action, and N,O-Diacetylmuramidase also can suppress the growth of adenovirus.In addition, N,O-Diacetylmuramidase has the effect of antiplasmin protein dissolution, participates in the reparation of gap conjunctive tissue and the biosynthesizing of mucopolysaccharide etc.
According to the present invention, soya-bean milk should be appreciated that it is that whole grain soybean process adds a kind of beverage that obtains after the steps such as water, squeezing, heating and filtration, and wherein the soybean solid substance should have more than 8%, but does not add any other composition.The processing soya-bean milk should be appreciated that and is to use whole grain soybean or skimmed soy beans; Adopt the resulting liquid of above-mentioned preparation method; Or add water with the powder soya-bean milk and process liquid, adding a kind of emulsus beverage that vegetables oil, sugar, salt and emulsifying agent etc. are processed again, its soybean solid substance should be more than 6%.
Concrete preparation method is following for soya-bean milk: soybean is flooded a night in water, reaches 2.2～2.3 times of original weight in its weight of back that fully absorbs water.The dipping soybean grinds with stone mortar or emery wheel mill etc., and is more carefully good more.Grinding fermented bean drink heats then and becomes soya-bean milk; Heating can make protein, fat and the stripping well of other composition in the soybean; Also make objectionable impurities passivation such as the loose material of trypsinase statin, red Archon hemoglutinin and Tiroidina, kill the mikrobe that is attached on the soybean etc.
Described recombined milk is with 120-150 weight part milk powder and the preparation of 850-880 weight part softening water.
In the present invention, described milk powder is extensive in the market product sold.
People know that water hardness mainly is made up of calcium (Ca2+), magnesium (Mg2+) ion.When the former water that contains calcium, mg ion passed through the interchanger resin layer, the calcium in the water, mg ion were discharged sodium ion simultaneously by resin absorption, are exactly the softening water that has removed water hardness ions from the effusive water of interchanger like this.After resin absorption calcium, mg ion reach certain saturation degree; The outflow water hardness increases; This moment, water softener can carry out the reproduction operation of exhausted resin automatically according to preset program, and the sodium chloride solution (salt solution) that utilizes higher concentration makes exhausted resin return to sodium type resin again through resin.Described water softener is general in the market product sold.
Described sugar is selected from sucrose, for example brown sugar, white sugar, granulated sugar, brown sugar; Glucose, fructose, semi-lactosi, lactose, SANMALT-S, starch, dextrin or glycogen marsh-mallow.
Preferably, the sugared emulsion that contains of 930-950 weight part sweet milk or recombined milk and 50-70 weight part sugar is heated to temperature 62-64 ℃.
More preferably, the sugared emulsion that contains of 940 weight part sweet milks or recombined milk and 60 weight parts sugar is heated to temperature 62-64 ℃.
Then, heating contain sugared emulsion temperature 60-65 ℃ with homogenization pressure 15-25MPa under carried out homogeneous 10-30 minute.
Homogeneous is technology of often using during food or chemical industry are produced.In food-processing, described homogeneous is meant that the feed liquid of material makes the material refinement under extruding, thump and decompression expansible triple role, mixes thereby material is reached more equably.For example in milk preparation processing, use the clarifixator must be littler, thereby make the whole prod system more stable fatty broken in the milk.Homogeneous mainly is to use clarifixator to realize that clarifixator is the important processing units in food, dairy products, the beverage industry.In the present invention, employed clarifixator is extensive in the market product sold, and for example the clarifixator of mechanical means ltd production and sales is closed according to willing mechanical means ltd, east, Shanghai magnificent high pressure homogenizer factory, Hang Zhouhui in Shanghai.
Preferably, said heating contain sugared emulsion temperature 62-65 ℃ with homogenization pressure 16-22MPa under homogenize and handle 14-26min.
More preferably, said heating contain sugared emulsion temperature 62-64 ℃ with homogenization pressure 18-20MPa under homogenize and handle 18-22min.
The homogeneous liquid that the obtains 5-15s that under temperature 120-140 ℃, sterilizes again.
Sterilization means the method for the mikrobe in the material being killed or removing with heating power or other proper method.In the present invention, described sterilising method is the method that adopts usually in the present technique field, for example moist heat sterilization, hot-air sterilization, filtration sterilization method etc.Moist heat sterilization means that material utilizes HP steam or other thermodynamics sterilization means kill bacteria in Sterilizers, and the sterilization ability is very strong, for the most effectively reaching the widest method of purposes in the thermodynamics sterilization.The present invention is employed in the moist heat sterilization under temperature 120-140 ℃.
Then, being cooled to temperature 37-42 ℃ through the sugared emulsion of handling that contains that homogenizes.Can adopt the various types of cooling to cool off, for example cool off, perhaps use the various cooling apparatuss of selling in the market to cool off with water coolant.Inoculate the 0.02-0.10 weight part and be described below 1 * 10 of cultivation
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs.
The cultural method of described lactobacterium helveticus H9 is following:
The preparation of substratum: measure the 943g deionized water, according to glucose 20.460g/L, soy peptone 10g/L, yeast powder 5g/L, Tryptones 10g/L, NaA
C6.835g/L, K
2HPO
42.929g/L, Trisodium Citrate 0.976g/L, MgSO
47H
2O 200.0mg/L, MnSO
45H
2O 54.0mg/L, L-halfcystine 0.25g/L, these compounds of Tween-80 1g/L weighing, then that they are miscible in its deionized water, prepare described substratum.
Culture condition and equipment: the nutrient solution that will connect bacterium places LRH-500F constant incubator (Shanghai Yiheng Scientific Instruments Co., Ltd) to cultivate 15-18 hour for 37 ℃.
In sweet milk or recombined milk, can also add the bacterial classification of delivering directly in said newborn gross weight 0.01-1.0% thermophilus streptococcus and lactobicillus bulgaricus.
The purpose of delivering directly bacterial classification that in sweet milk or recombined milk, adds thermophilus streptococcus and lactobicillus bulgaricus is to produce the fermented-milk that is different from single bacterium fermented-milk organoleptics property.Adding described thermophilus streptococcus and lactobicillus bulgaricus for example is the throw type leaven YC-X11 that is sold by Hansen Corp. of Denmark section.
Then, what contain lactobacterium helveticus H9 bacterial classification contains sugared emulsion at temperature 37-42 ℃ of bottom fermentation 7-8h, with inorganic base aqueous solution with the pH regulator of its fermented liquid to 4.2-4.6, at last at temperature 4-8 ℃ of following after-ripening 8-12h, obtain described lactobacterium helveticus H9 fermented-milk.
Described mineral alkali is selected from Pottasium Hydroxide, sodium hydroxide or yellow soda ash.The concentration of described inorganic base aqueous solution generally is 1-5N.The pH value of its fermented liquid can use people's normally used pH meter in the laboratory to measure, and also can be employed in the upward common pH value detection method that adopts of industry and detect.
The detection method that adopts this specification sheets to describe below has detected that lactobacterium helveticus H9 content is 2 * 10 in fermented-milk of the present invention
7Cfu/g-1 * 10
8Cfu/g.
In the after-ripening step of fermented-milk or in the fermented-milk that is obtaining, add in said fermented-milk gross weight 0.5-1.0% thickening material or essence.
On meaning of the present invention, described thickening material should be appreciated that it is that a kind of fermented-milk viscosity that can make increases, and gives the agent of fermented-milk with sticking sliding agreeable to the taste tongue sense, and every material with this performance all is operable usually.Described thickening material for example is selected from Danisco (China) ltd with trade(brand)name GRINDSTED SB 252-C product sold, or Beijing section opens up permanent logical bio tech ltd with trade(brand)name yoghourt stabilizer KY-309 product sold.
Described essence should be appreciated that it is a kind of material that can improve or strengthen fermented-milk fragrance and fragrance, and every material with this performance all is operable usually.Described essence for example is selected from Ai Ersen (Tianjin) essence and flavoring agent ltd with trade(brand)name natural yoghourt essence EF-4016 product sold, or Guangzhou Flower Flavours and Fragrances Co., Ltd. is with trade(brand)name sour milk essence MH-5061 product sold.
Through following experimental verification, fermented-milk of the present invention has Zinc metallopeptidase Zace1 (Angiotensin Converting Enzyme I) to be suppressed active and is rich in biologically active peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP).
It is following at temperature 4 ℃ of following storage period viable counts, pH value, ACE inhibiting rate and VPP and IPP content TP to contain lactobacterium helveticus H9 fermentation cow's milk of the present invention, horse breast and soya-bean milk:
1, lactobacterium helveticus H9 viable count measuring method:
Fermented liquid to certain multiple, adopts the dull and stereotyped tilt-pour process of MRS nutrient agar with the sterile saline gradient dilution, after 37 ℃ of temperature are cultivated 48h down, counts total number of bacterial colony.
2, pH values determination method:
Adopt accurate pH meter (SJ-3F type pH meter (Shanghai Precision & Scientific Instrument Co., Ltd. Leici Instrument Factory)) to measure fermented-milk pH value.
3, ACE inhibiting rate measuring method and data processing method:
A. with the above-mentioned fermented-milk for preparing centrifugal 10min under the 8000g condition, get supernatant, and with 10mol/LNaOH with its pH regulator to 8.3, centrifugal 10min under the 8000g condition again, the supernatant that obtains suppresses the active test sample of treating for measuring ACE.
B. hippuryl histidyl-leucine (HHL) and Zinc metallopeptidase Zace1 (ACE) all are dissolved in the 0.1mol/L sodium borate buffer solution (pH8.3) that contains 0.3mol/L NaCl.Fully shake mixing after in the 1.5mL centrifuge tube, adding 50L HHL solution (0.010mol/L) and the above-mentioned supernatant of 50 μ L; Under 37 ℃ of temperature, hatch 2min; Add 50 μ L ACE (0.010U/mL) solution then; Fully the concussion mixing reacts 30min down for 37 ℃ in temperature, makes the enzyme deactivation termination reaction in 85 ℃ of heating in water bath 10min of temperature again.And the urobenzoic acid content in the assaying reaction liquid.
C. urobenzoic acid assay: adopt urobenzoic acid (Hippruic acid, HA) content in the RP-HPLC method assaying reaction liquid.Chromatographic column: ZORBAX C18 (4.6 * 250mm, 5 μ m, Agilent, USA); Moving phase: volume ratio is 22% acetonitrile (containing 0.1% trifluoroacetic acid (TFA), volume ratio) and 78% deionized water (containing 0.1%TFA, volume ratio); Flow velocity: 1.5mL/min; Detect wavelength: 228nm; Column temperature: 30 ℃; Sample size: 20 μ L.
The ACE inhibiting rate calculates by following formula:
ACE inhibiting rate (%)=([HA] c-[HA] s)/([HA] c-[HA] h) * 100%
In the formula:
[HA] c is a control sample urobenzoic acid concentration (adding deionized water);
[HA] s is a testing sample urobenzoic acid concentration;
[HA] h is a urobenzoic acid concentration in the HHL standard substance.
4, VPP and IPP content assaying method:
Adopt GC-MS (LC-ESI-MS/MS) to measure.Fermented-milk is centrifugal 10min under the 8000g condition, gets supernatant, and with 10M NaOH with its pH regulator to 8.3, centrifugal 10min under the 8000g condition again, the supernatant that obtains is the testing sample of VPP and IPP content.
HPLC condition: chromatographic column, CAPCELL PAK C
18(MG, 2.0 * 100mm, 5 μ m, Shiaeibo, Japan); Flow velocity, 0.2mL/min; Detect wavelength: 210nm; Column temperature: 30 ℃; Sample size: 10 μ L.Mobile phase A is the deionized water that contains by volume 0.1%TFA; Mobile phase B is the deionized water that contains by volume 0.1% trifluoroacetic acid acetonitrile; Gradient elution: in 0min (95% mobile phase A+5% Mobile phase B); In 25min (65% mobile phase A+35% Mobile phase B); 25.5min (Mobile phase B is 100%); 35min (Mobile phase B is 100%); In 35.5min (95% mobile phase A+5% Mobile phase B); In 45min (95% mobile phase A+5% Mobile phase B).
ESI-MS/MS condition: POSITIVE MODE, Shealth gas flow rate:35arb; Aux/sweep gas flow rate:5arb; I spray voltage:5.50Kv; Capillary temp:300 ℃; Capillary voltage:16Kv; The secondary collision energy is 30%; The fragment sweep limit is 100-400m/z.
Accompanying drawing 1-4 lists respectively and contains the variation at temperature 4 ℃ of following storage period viable counts, pH value, ACE inhibiting rate and VPP and IPP content of lactobacterium helveticus H9 fermentation cow's milk of the present invention, horse breast and soya-bean milk.
Clearly illustrate that by these results; Containing lactobacterium helveticus H9 fermentation cow's milk of the present invention, horse breast and soya-bean milk stored 1-28 days down for 4 ℃ in temperature; The viable count of lactobacterium helveticus H9 of the present invention (cow's milk: 7.3-8.1 logCFU/g) remain unchanged basically with pH value (4.2-4.4); Therefore, contain lactobacterium helveticus H9 fermentation cow's milk of the present invention, horse breast and soya-bean milk and have storage stability preferably.
The ACE inhibiting rate that contains lactobacterium helveticus H9 fermentation cow's milk, horse breast and soya-bean milk of the present invention can reach 75%, 65% and 63% respectively; The report of the ACE inhibiting rate of the fermented-milk of not seeing on the home market to be sold.
The VPP and the IPP content that contain lactobacterium helveticus H9 fermentation cow's milk of the present invention can reach 3.0-3.3 μ M and 2.2-2.5 μ M respectively, and do not find to contain the fermented milk prod of VPP and IPP on the home market.
[beneficial effect]
Beneficial effect of the present invention is following:
The present invention is inoculation lactobacterium helveticus H9 bacterial classification in sweet milk (cow's milk, horse breast, soya-bean milk) or recombined milk, makes the lactobacterium helveticus H9 in the fermented-milk of the present invention keep very high viable count (＞10
7Cfu/g), fermented-milk of the present invention has ACE inhibition activity and is rich in biologically active peptides Val-Pro-Pro (VPP) and Ile-Pro-Pro (IPP).Therefore, fermented-milk of the present invention have bring high blood pressure down, functional performance such as preventing cardiovascular disease and the intravital micro-ecological environment of improvement machine.
Lactobacterium helveticus (Lactobacillus helveticus) H9 on April 28th, 2011 in common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms (CGMCC) preservation, preserving number is CGMCC4811.
[description of drawings]
Fig. 1 representes the variation at 4 ℃ of following storage period viable counts of temperature of lactobacterium helveticus H9 fermentation cow's milk of the present invention, horse breast and soya-bean milk.
Fig. 2 representes the variation in 4 ℃ of following storage period pH values of temperature of lactobacterium helveticus H9 fermentation cow's milk, horse breast and soya-bean milk.
Fig. 3 representes the variation at 4 ℃ of following storage period ACE inhibiting rates of temperature of lactobacterium helveticus H9 fermentation cow's milk, horse breast and soya-bean milk.
Fig. 4 representes the variation of lactobacterium helveticus H9 fermentation cow's milk at 4 ℃ of following storage period VPP of temperature and IPP content.
[embodiment]
To understand the present invention better through non-limiting example below.
Embodiment 1: the present invention contains the preparation of lactobacterium helveticus H9 fermentation cow's milk
Preparation process is following:
In 920 weight part fresh cow milks, add 40 weight parts sugar; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 60 ℃ of temperature, and the clarifixator that uses Shanghai east magnificent high pressure homogenizer factory to produce then carried out homogeneous 10 minutes under homogenization pressure 15MPa, the homogeneous liquid that the obtains 5s that under 120 ℃ of temperature, sterilizes again; Be cooled to 37 ℃ of temperature with tap water then, 0.02 weight part 1 * 10 that obtains is cultivated in inoculation according to cultural method that this specification sheets is described
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs, and then at 37 ℃ of bottom fermentation 7.0h of temperature, with the pH regulator to 4.2 of 1N potassium hydroxide aqueous solution with its fermented liquid, at 4 ℃ of following after-ripening 8h of temperature, obtains containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 1.0 * 10
8Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 75%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.2 μ M and 2.3 μ M.
Embodiment 2: the present invention contains the preparation of lactobacterium helveticus H9 fermentation horse breast
Preparation process is following:
Add 50 weight parts sugar toward the bright horse of 950 weight parts Ruzhong; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 61 ℃ of temperature, and the clarifixator that uses Shanghai east magnificent high pressure homogenizer factory to produce then carried out homogeneous 26 minutes under homogenization pressure 24MPa, the homogeneous liquid that the obtains 12s that under 125 ℃ of temperature, sterilizes again; Be cooled to 41 ℃ of temperature with tap water then, 0.05 weight part 1.5 * 10 that obtains is cultivated in inoculation according to cultural method that this specification sheets is described
11Cfu/g lactobacterium helveticus H9 bacterial classification; Stir, then at 41 ℃ of bottom fermentation 7.4h of temperature, with the pH regulator to 4.4 of 1N mineral alkali aqueous sodium hydroxide solution its fermented liquid; At 6 ℃ of following after-ripening 9h of temperature, obtain containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 1.5 * 10
7Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 65%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.1 μ M and 2.4 μ M.
Embodiment 3: the present invention contains the preparation of lactobacterium helveticus H9 fermentation horse breast
Preparation process is following:
Add 60 weight parts sugar toward the bright horse of 940 weight parts Ruzhong; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 64 ℃ of temperature, and the clarifixator that uses Shanghai east magnificent high pressure homogenizer factory to produce then carried out homogeneous 24 minutes under homogenization pressure 18MPa, the homogeneous liquid that the obtains 8s that under 130 ℃ of temperature, sterilizes again; Be cooled to 38 ℃ of temperature with tap water then, 0.04 weight part 1.6 * 10 that obtains is cultivated in inoculation according to cultural method that this specification sheets is described
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs, and then at 38 ℃ of bottom fermentation 7.2h of temperature, with the pH regulator to 4.4 of 1N aqueous sodium carbonate with its fermented liquid, at 5 ℃ of following after-ripening 10h of temperature, obtains containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 2.0 * 10
7Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 63%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.3 μ M and 2.5 μ M.
Embodiment 4: the present invention contains the preparation of lactobacterium helveticus H9 fermentation cow's milk
Preparation process is following:
In 930 weight part fresh cow milks, add 70 weight parts sugar; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 63 ℃ of temperature, and the clarifixator that uses Shanghai east magnificent high pressure homogenizer factory to produce then carried out homogeneous 20 minutes under homogenization pressure 22MPa, the homogeneous liquid that the obtains 6s that under 135 ℃ of temperature, sterilizes again; Be cooled to 40 ℃ of temperature with tap water then, 0.08 weight part 1 * 10 that obtains is cultivated in inoculation according to cultural method that this specification sheets is described
11Cfu/g lactobacterium helveticus H9 bacterial classification; Stir, then at 40 ℃ of bottom fermentation 7.6h of temperature, with the pH regulator to 4.3 of 2N mineral alkali aqueous sodium hydroxide solution its fermented liquid; At 6 ℃ of following after-ripening 11h of temperature, obtain containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 2.6 * 10
7Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 72%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.0 μ M and 2.2 μ M.
Embodiment 5: the present invention contains the preparation of lactobacterium helveticus H9 fermentation recombined milk
Preparation process is following:
In the recombined milk of 940 weight parts, add 50 weight parts sugar by 120 weight part milk powder and the preparation of 880 weight part softening waters; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 62 ℃ of temperature; The clarifixator that uses east, Shanghai magnificent high pressure homogenizer factory to produce then carried out homogeneous 15 minutes under homogenization pressure 20MPa; The homogeneous liquid that the obtains 10s that under 138 ℃ of temperature, sterilizes again is cooled to 39 ℃ of temperature with tap water, inoculation 0.06 weight part 2 * 10 that cultivation obtains according to cultural method that this specification sheets is described then
11Cfu/g lactobacterium helveticus H9 bacterial classification; Stir, then at 39 ℃ of bottom fermentation 7.8h of temperature, with the pH regulator to 4.5 of 1N mineral alkali aqueous sodium hydroxide solution its fermented liquid; At 7 ℃ of following after-ripening 10h of temperature, obtain containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 2.0 * 10
7Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 75%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.2 μ M and 2.3 μ M.
Embodiment 6: the present invention contains the preparation of lactobacterium helveticus H9 fermentation recombined milk
Preparation process is following:
In the recombined milk of 960 weight parts, 150 weight part milk powder and the preparation of 850 weight part softening waters, add 80 weight parts sugar; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to 65 ℃ of temperature; The clarifixator that uses east, Shanghai magnificent high pressure homogenizer factory to produce then carried out homogeneous 30 minutes under homogenization pressure 25MPa; The homogeneous liquid that the obtains 15s that under 140 ℃ of temperature, sterilizes again is cooled to 42 ℃ of temperature with tap water, inoculation 0.10 weight part 1 * 10 that cultivation obtains according to cultural method that this specification sheets is described then
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs, and then at 42 ℃ of bottom fermentation 8.0h of temperature, with the pH regulator to 4.6 of 1N mineral alkali aqueous sodium carbonate with its fermented liquid, at 8 ℃ of following after-ripening 12h of temperature, obtains containing described lactobacterium helveticus H9 fermented-milk at last.Adopt the described method of this specification sheets to measure and obtain, it contains 3.0 * 10
7Cfu/g lactobacterium helveticus H9, its ACE suppresses activity 73%, and the content of biologically active peptides Val-Pro-Pro and Ile-Pro-Pro is respectively 3.1 μ M and 2.2 μ M.
Test example 1: the present invention contains the clinical trial of lactobacterium helveticus H9 fermentation cow's milk
The clinical trial case is selected:
1, meets the hypertension Case definition and can include the test case in.
2, experimenter's physical appearance is fair, does not have other disease.
3, the age is at 18-65 between year, non-gestation or lactating women, non-allergic constitution.
Get rid of the case standard:
Be associated with serious primary disease such as cardiovascular, the cerebrovascular, liver, kidney and hemopoietic system, the mental patient.
TP:
1, adopts randomized blocks
2, observe 20 examples
3, drinking method: drink the fermentation cow's milk of the embodiment of the invention 1 preparation, each 100ml, every day 3 times.
4, test period: 3 months, do not carry out other treatment relevant during the clinical verification with this disease.
Curative effect determinate standard:
1, produce effects: the blood pressure depreciation and continues more than 1 month more than 10mmHg.
2, effective: the blood pressure depreciation is at 5-10mmHg, and continues more than 1 month.
3, invalid: blood pressure does not have considerable change.
Test-results:
Table 1: blood pressure test result analysis table
| Criterion of therapeutical effect | The example number | Produce effects, example | Effectively, example | Invalid, example | Efficient, % |
| The |
10 | 4 | 4 | 2 | 80 |
| |
10 | 0 | 1 | 9 | 10 |
Control sample is according to State Standard of the People's Republic of China GB19302-2010, the fermented-milk of food safety national standard preparation.
Above-mentioned test-results shows that the present invention contains lactobacterium helveticus H9 fermentation cow's milk and has the effect that obviously brings high blood pressure down.
Claims (10)
1. a lactobacterium helveticus (Lactobacillus helveticus) H9, this bacteria strain on April 28th, 2011 in the preservation of common micro-organisms DSMZ of China Committee for Culture Collection of Microorganisms, preserving number is CGMCC4811.
2. the purposes of lactobacterium helveticus H9 according to claim 1 in the preparation fermented-milk.
3. purposes according to claim 2 is characterized in that the preparation process of the said fermented-milk that contains lactobacterium helveticus H9 is following:
In 920-960 weight part sweet milk or recombined milk, add 40-80 weight part sugar; Remove impurity at sugar dissolving after-filtration; Then it is contained sugared emulsion is heated to temperature 60-65 ℃, under homogenization pressure 15-25MPa, carries out the homogeneous liquid that the obtains 5-15s that under temperature 120-140 ℃, sterilizes again then homogeneous 10-30 minute; Be cooled to temperature 37-42 ℃ then, inoculation 0.02-0.10 weight part 1 * 10
11Cfu/g lactobacterium helveticus H9 bacterial classification stirs, then at temperature 37-42 ℃ of bottom fermentation 7-8h, with inorganic base aqueous solution with the pH regulator of its fermented liquid to 4.2-4.6, at last at temperature 4-8 ℃ of following after-ripening 8-12h, obtain described lactobacterium helveticus H9 fermented-milk.
4. purposes according to claim 3 is characterized in that described sweet milk is selected from cow's milk, horse breast or soya-bean milk.
5. purposes according to claim 3 is characterized in that described recombined milk is with 120-150 weight part milk powder and the preparation of 850-880 weight part softening water.
6. purposes according to claim 3 is characterized in that described sugar is selected from sucrose, glucose, fructose, semi-lactosi, lactose, SANMALT-S, starch, dextrin or glycogen marsh-mallow.
7. purposes according to claim 3 is characterized in that described mineral alkali is selected from Pottasium Hydroxide, sodium hydroxide or yellow soda ash.
8. purposes according to claim 3 is characterized in that in sweet milk or recombined milk, adding the bacterial classification of delivering directly in said newborn gross weight 0.01-1.0% thermophilus streptococcus and lactobicillus bulgaricus.
9. purposes according to claim 3 is characterized in that described fermented-milk contains 2 * 10
7Cfu/g-1 * 10
8Cfu/g lactobacterium helveticus H9, the content of VPP are 3.0-3.3 μ M, and the content of IPP is 2.2-2.5 μ M.
10. purposes according to claim 3 adds in said fermented-milk gross weight 0.5-1.0% thickening material or essence in the fermented-milk that it is characterized in that in the after-ripening step of fermented-milk, perhaps obtaining.
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| CN106261363A (en) * | 2016-08-08 | 2017-01-04 | 厦门和美科盛生物技术有限公司 | A kind of probiotic bacteria fruit and vegerable enzyme beverage and preparation method thereof |
| CN108949630A (en) * | 2018-07-27 | 2018-12-07 | 内蒙古农业大学 | A kind of koumiss leavening and its preparation method and application |
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| CN111979286A (en) * | 2020-08-31 | 2020-11-24 | 鲁东大学 | Method for preparing shellfish high F value oligopeptide by combining fermentation method and enzyme method |
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| CN102329762A (en) * | 2011-11-01 | 2012-01-25 | 内蒙古农业大学 | Lactobacillus helveticus H9 and application thereof |
| CN102960444A (en) * | 2012-11-19 | 2013-03-13 | 陕西科技大学 | Preparation method of goat milk containing ACE (Angiotensin Converting Enzyme) inhibitory peptide based on Lactobacillus helveticus fermentation |
| CN103141575A (en) * | 2013-03-15 | 2013-06-12 | 南京师范大学 | Method for preparing hawthorn yogurt being rich in angiotensin-converting enzyme inhibition peptides |
| CN106261363A (en) * | 2016-08-08 | 2017-01-04 | 厦门和美科盛生物技术有限公司 | A kind of probiotic bacteria fruit and vegerable enzyme beverage and preparation method thereof |
| CN108949630A (en) * | 2018-07-27 | 2018-12-07 | 内蒙古农业大学 | A kind of koumiss leavening and its preparation method and application |
| CN108949630B (en) * | 2018-07-27 | 2020-06-30 | 内蒙古农业大学 | Fermented mare milk leavening agent and preparation method and application thereof |
| CN109517763A (en) * | 2018-12-27 | 2019-03-26 | 内蒙古农业大学 | Lactobacillus helveticus H11 and its application |
| CN109517763B (en) * | 2018-12-27 | 2022-03-22 | 内蒙古农业大学 | Lactobacillus helveticus H11 and application thereof |
| CN111979286A (en) * | 2020-08-31 | 2020-11-24 | 鲁东大学 | Method for preparing shellfish high F value oligopeptide by combining fermentation method and enzyme method |
| CN111979286B (en) * | 2020-08-31 | 2021-11-26 | 鲁东大学 | Method for preparing shellfish high F value oligopeptide by combining fermentation method and enzyme method |
| CN114903083A (en) * | 2022-06-09 | 2022-08-16 | 陕西科技大学 | Biological active peptide probiotic fermented sheep milk and preparation method thereof |
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