CN106900856A - The balloonflower root fermented ginseng Yoghourt and its production method of a kind of green - Google Patents
The balloonflower root fermented ginseng Yoghourt and its production method of a kind of green Download PDFInfo
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Abstract
The present invention relates to the balloonflower root fermented ginseng Yoghourt and its production method of a kind of green, belong to nutraceutical processing technique field.Extract the active ingredient in balloonflower root and ginseng respectively by supercritical fluid extraction, ginseng is fermented using beta-glucosidase then, make rare ginsenoside Rd, PPD, Rg in balloonflower root ginseng Yoghourt3, Rh2Content substantially increase, then by the fermentation of streptococcus thermophilus and lactobacillus bulgaricus, obtain pure plant balloonflower root fermented ginseng Yoghourt.The present invention is especially suitable for the elderly without additives such as preservative, sweetener, thickener, pigments, can improve the health care of sleep, enhancing cardiac function, improves the symptom of shortness of breath uncomfortable in chest, expelling phlegm and arresting coughing, and body's immunity can be significantly increased, take supercritical fluid extraction this " Green Chemical Technology ", efficiently, cleaning, process is simple, recovery rate is higher, and solvent safety is easily reclaimed, Product Green is pollution-free, is a kind of new pure plant, environmentally friendly nutraceutical.
Description
Technical field
The invention belongs to nutraceutical processing technique field, refer in particular to a kind of green balloonflower root fermented ginseng Yoghourt and its
Production method.
Background technology
Yoghourt (yogurt, yoghurt), be one kind with milk as raw material, by after pasteurize again to milk in be added with
Beneficial lactic acid bacteria, the filling milk product of fermented cooling.Yoghourt not only remains the advantage of milk, and taste is sour-sweet fine and smooth,
Containing biodiasmin, processed process is also maximized favourable factors and minimized unfavourable ones in some aspects, as the nourishing and health food for being more suitable for the mankind
Product.The many fruit-flavor types with auxiliary materials such as coagulating type, stirred type and the various fruit juice jams of addition of sour milk product are many in the market.Acid
The lactic acid bacteria contained in milk is a kind of probiotics, through the beneficial health of the Yoghourt after lactobacillus-fermented, in can making milk
The macromolecular substances such as sugar, protein are hydrolyzed into lactose, lactic acid, small peptide chain and amino acid etc., it is easier to be absorbed by organisms；Energy
Enough promote intestines peristalsis, promote thalline raised growth to change osmotic pressure, so as to promote the digestion to prevent constipation；It is able to maintain that enterobacteriaceae
Group's ecological balance, forms biological barrier, suppresses invasion of the harmful bacteria to enteron aisle, suppresses growth of the saprophytic bacteria in enteron aisle, suppresses rotten
Produced toxin is lost, the generation of the harmful substances such as ammonia in its metabolin, hydrogen sulfide and scatol is reduced, therefore has anti-ageing
Always, the effect of pigment deposition is prevented, makes liver and brain from harm；The material of enhancing immunologic function can be produced, people is improved
Body immunity；Lactic acid bacteria can also produce multivitamin necessary to human nutrition during the fermentation, such as VB1, VB2, VB6,
VB12 etc.；The lactic acid produced after fermentation can effectively improve utilization rate of the mineral matters such as calcium, phosphorus in human body, therefore the calcium in Yoghourt
Phosphorus is easier to be absorbed by the body；And the crowd bad to lactose digestion, eating Yoghourt will not also occur abdominal distension, gas more or suffer from diarrhoea existing
As.With the continuous improvement of living standards of the people, the enhancing of health care consciousness, people more focus on healthcare function and the nutrition of diet
Property, turn into a kind of development trend to some nutrition classes or the new Yoghourt of functional raw material production are added in Yoghourt.
Balloonflower root (Platycodon grandiflorus) is Campanulaceae (Campanulaceae) Platycodon grandiflouorum
(Platycodon) herbaceos perennial, bitter in taste, pungent, flat, return lung warp, with facilitaing lung relieving sore-throat, the effect of eliminating the phlegm apocenosis.With
It is uncomfortable in chest not smooth in coughing with a lot of sputum, pharyngalgia, the disease such as lung carbuncle pyemesis.Balloonflower root is China and the important medicine-food two-purpose of other Asian countries
Resource, is not only a kind of traditional Chinese medicine, and the application in Chinese medicine compound prescription is wider, but also is a kind of food.Balloonflower root is nutritious,
So far, nutritional ingredient isolated from balloonflower root and function factor are main based on triterpene saponin, flavonoids, also contain
There are polysaccharide, volatile oil, steroidal, several amino acids and various micro elements needed by human.Modern pharmacology research shows, balloonflower root
With anti-inflammatory, eliminating the phlegm, antiulcer, hypotensive, expansion of blood vessels, antipyretic-antalgic, calmness, hypoglycemic, cholinolytic, promotion bile point
Secrete, antiallergy and the extensive pharmacological action of enhancing body immunity etc..Ginseng (Panax ginseng C.A.Meyer) is slender acanthopanax
Section (Araliacede) Panax (Panax) plant, mainly originates in Northeast China and the Korea peninsula, have the U.S. of " kings of hundred grass "
Reputation.The earliest medical masterpiece of China《Sheng Nong's herbal classic》Describe：Ginseng has that " main tonifying five zang organs, soothe the nerves, determine soul, and stop palpitate with fear is removed
Perverse trend, eyesight-improving intelligence-developing, long term usage is made light of one's life by commiting suicide macrobiosis ".Tool reinforces vital energy, veins takes off admittedly, reinforce the spleen to benefit the lung, the function such as promoting production of body fluid and inducing sedation of the mind.2012
Year China's approval ginseng becomes medicine-food two-purpose natural resources as new resource food, is that extensive utilize of the ginseng in food carries
Supply to ensure.Ginseng contains various nutrition and health-care components, including ginsenoside, polysaccharide, protein, amino acid, flavones (different Huang
Ketone), inorganic elements, vitamin, organic acid, alkaloid, aliphatic acid, sterol, nucleosides, lignin and volatile oil etc..Modern pharmacology
Learn research to show, ginseng has antitumor, regulation central nervous system, regulation immune system, regulation blood sugar, the myocardium work(of increase
The multiple pharmacological activity effect such as energy, anti-hemolysis, anti-aging, antifatigue.The main active of ginseng is ginsenoside, main point
It is protopanoxadiol, Protopanaxatriol and oleanane type ginsenoside.Rb1, Rg1, Re equal size in natural ginsenoside
It is higher, and the rare saponin content such as Rd, Rh1, Rh2, Compound K is less or does not exist.Many pharmacological researches show, rare
Ginsenoside drug effect is more precious, cardiovascular and cerebrovascular, nervous system, anti-good fortune are penetrated, anti-inflammatory, the effect such as antitumor it is unique.
Supercritical fluid extraction (Supercritical Fluid Extraction, abbreviation SPE), is to utilize shooting flow
The solvation effect of body dissolves liquid or solid mixture to be separated, then reduces shooting flow by decompression or temperature regulating
The density of body, so as to reduce its solvent power, is separated extract.It is reported that using supercritical CO2Abstraction technique is extracted
The nutritional ingredients such as the ginsenoside in ginseng, have the advantages that separating technology is simple, environment-friendly, protect heat-sensitive substance.
Beta-glucosidase (β-glucosidase EC 3.2.1.21), belongs to hydrolase, also known as β-D-Glucose glycosides water
Solution enzyme, gentiobiase, cellobiase, are to be obtained to form through liquid deep layer fermenting purification by fungi strain.
The content of the invention
The present invention provides the balloonflower root fermented ginseng Yoghourt and its production method of a kind of green, general to solve current fermented yoghourt
Store-through the problem that nutritional ingredient is not enough and production technology falls behind.
The present invention is adopted the technical scheme that, obtained by the following steps：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 4~12%, ginseng 8~16%, working stock culture 5~
10%, surplus fresh milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 1:2~
2:1；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of granularity 60-80 mesh is ground into；Ginseng is taken, the ginseng pulverate of granularity 80-100 mesh is ground into
End；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 2~5 times of entrainers are added, is placed in supercritical extracting equipment, 10~12h, extraction are soaked at room temperature
The time is taken for 3~5h, number of times is 2~4 times, temperature is 30~50 DEG C, pressure is 20~40MPa, CO2Flow velocity keeps 12L/h not
Become, collect product in separating still and obtain balloonflower root extraction powder；Ginseng Root Powder is taken, 2~5 times of entrainers are added, supercritical extract is placed in and is set
In standby, 20~24h is soaked at room temperature, extraction time is 3~5h, and number of times is 2~4 times, and temperature is 30~50 DEG C, pressure is 20~
40MPa, CO2Flow velocity keeps 15L/h constant, collects product in separating still and obtains ginseng extraction powder；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, beta-glucosidase used
Enzyme, performs Q/NXM010 standards, meets GB25594-2009《National food safety standard food industry enzyme preparation》Standard, will
Ginseng extracts powder, carries out sterilization treatment, adds 1~3 times of amount pure water, accesses beta-glucosidase, is put into constant incubator,
3~5d is cultivated in pH value 4.0~5.0, temperature 50 C~60 DEG C, and terminating reaction by zymotic fluid suction filtration, extraction, is reclaimed, sent out
Ginseng extraction powder after ferment；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C is carried out at degreasing
Reason；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh ox
Milk is well mixed, and stirs 10~15min, and being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
Take the homogenate that step (6) is obtained, add the working stock culture in formula, ferment 3 under the conditions of being placed in 35~45 DEG C~
5h, it is seen that the upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root
Fermented ginseng Yoghourt, refrigerates under the conditions of being subsequently placed in 2-5 DEG C.
Entrainer is 70% ethanol solution in step (3) of the present invention.
Supercritical fluid extraction is as a kind of new " green " extraction and separation technology with broad prospect of application at present
The advanced technology of current food processing industry.The Yoghourt is especially suitable for the elderly, can improve the health care of sleep, and strengthens cardiac function, changes
It is apt to the symptom of shortness of breath uncomfortable in chest, expelling phlegm and arresting coughing.
The main active of ginseng is ginsenoside, is broadly divided into protopanoxadiol, Protopanaxatriol and oleanane
Type ginsenoside.Rb1, Rg1, Re equal size are higher in natural ginsenoside, and Rd, Rh1、Rh2, PPD, Compound K etc.
Rare saponin content is less or does not exist.Many pharmacological researches show that rare ginsenoside drug effect is more precious, to heart and brain blood
Pipe, nervous system, anti-good fortune are penetrated, anti-inflammatory, the effect such as antitumor are unique.Rd has anti-arrhythmia, antiviral, expansion of blood vessels, resists
The functions such as oxidation, strengthen immunity；Rh2 has cancer cell specific induction of apoptosis, suppresses growth of tumour cell and transfer, and anti-inflammatory resists
Allergy, anti-dementia etc.；Rg3 has antitumor activity, improves the effect such as insulin signaling, reducing blood lipid, antithrombotic, antianxiety, with
Chemotherapeutics is used in conjunction with that immunity of organisms can be improved；PPD has antitumor, the effect such as strengthen immunity.
The present invention obtains the active ingredient in balloonflower root and ginseng using supercritical fluid extraction, environment-friendly pollution-free,
And extraction yield is higher, ginseng is fermented using beta-glucosidase, makes rare ginsenoside Rd in balloonflower root ginseng Yoghourt,
PPD, Rg3, Rh2Content substantially increase.The balloonflower root ginseng Yoghourt for obtaining, is especially suitable for the elderly, can improve the health care of sleep, and increases
Strong cardiac function, improves the symptom of shortness of breath uncomfortable in chest, expelling phlegm and arresting coughing, and can significantly increase body's immunity.
The present invention takes supercritical fluid extraction this " green without additives such as preservative, sweetener, thickener, pigments
Colour chemistry technology ", efficiently, cleaning, process is simple, recovery rate is higher, and solvent safety is easily reclaimed, and Product Green is pollution-free, is one
Plant new pure plant, environmentally friendly nutraceutical.
Brief description of the drawings
Fig. 1 is 19 kinds of ginsenoside reference substance HPLC spectrograms, in figure：
1：Rg1；2：Re；3：Rf；4：Rh1；5：Rg2；6：Rb1；7：Rc；8：Ro；9：F1；10：Rb2；11：Rb3；12：Rd；
13：20(S)-Rg3；14：20(R)-Rg3；15：PPT；16：F2；17：Rh2；18：Oleanolic acid；19：PPD；
Fig. 2 is the HPLC spectrograms through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation, in figure：
1：Rd；2：20(R)-Rg3；3：Rh2；4：PPD；
Fig. 3 is the HPLC spectrograms without the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation, in figure：
1：Rd；2：20(R)-Rg3；3：Rh2；4：PPD.
Specific embodiment
Embodiment 1
Obtained by the following steps：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 4%, ginseng 8%, working stock culture 5%, the fresh ox of surplus
Milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 1:2；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of the mesh of granularity 60 is ground into；Ginseng is taken, the Ginseng Root Powder of the mesh of granularity 80 is ground into；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 2 times of entrainers are added, the entrainer is 70% ethanol solution, is placed in supercritical extracting equipment
In, 10h is soaked at room temperature, extraction time is 3h, and number of times is 2 times, and temperature is 30 DEG C, and pressure is 20MPa, CO2Flow velocity keeps
12L/h is constant, collects product in separating still and obtains balloonflower root extraction powder；Ginseng Root Powder is taken, 2 times of entrainers are added, overcritical extraction is placed in
In taking equipment, 20h is soaked at room temperature, extraction time is 3h, and number of times is 2 times, and temperature is 30 DEG C, and pressure is 20MPa, CO2Flow velocity
Keep 15L/h constant, collect product in separating still and obtain ginseng extraction powder；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, beta-glucosidase used
Enzyme, performs Q/NXM010 standards, meets GB25594-2009《National food safety standard food industry enzyme preparation》Standard, by
Suzhou Kunlan Biotechnology Co., Ltd. is provided, and ginseng is extracted into powder, carries out sterilization treatment, adds 1 times of amount pure water, access β-
Glucuroide, is put into constant incubator, pH value 4.0, temperature 50 C, cultivates 3d, terminating reaction, by zymotic fluid suction filtration, extraction
Take, reclaim, the ginseng extraction powder after being fermented；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C is carried out at degreasing
Reason；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh ox
Milk is well mixed, and stirs 10min, and being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
The homogenate that step (6) is obtained is taken, the working stock culture in formula is added, ferments 3h under the conditions of being placed in 35 DEG C, it is seen that
The upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root ginseng hair
Ferment Yoghourt, refrigerates under the conditions of being subsequently placed in 2 DEG C.
Embodiment 2
Obtained by the following steps：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 8%, ginseng 12%, working stock culture 8%, the fresh ox of surplus
Milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 1:1；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of the mesh of granularity 80 is ground into；Ginseng is taken, the Ginseng Root Powder of the mesh of granularity 100 is ground into；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 3.5 times of entrainers are added, the entrainer is 70% ethanol solution, is placed in supercritical extract and sets
In standby, 11h is soaked at room temperature, extraction time is 4h, and number of times is 3 times, and temperature is 40 DEG C, and pressure is 30MPa, CO2Flow velocity keeps
12L/h is constant, collects product in separating still and obtains balloonflower root extraction powder；Ginseng Root Powder is taken, 4 times of entrainers are added, overcritical extraction is placed in
In taking equipment, 22h is soaked at room temperature, extraction time is 4h, and number of times is 3 times, and temperature is 40 DEG C, and pressure is 30MPa, CO2 flow velocitys
Keep 15L/h constant, collect product in separating still and obtain ginseng extraction powder；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, beta-glucosidase used
Enzyme, performs Q/NXM010 standards, meets GB25594-2009《National food safety standard food industry enzyme preparation》Standard, by
Suzhou Kunlan Biotechnology Co., Ltd. is provided, and ginseng is extracted into powder, carries out sterilization treatment, adds 2 times of amount pure water, access β-
Glucuroide, is put into constant incubator, pH value 4.5,55 DEG C of temperature, cultivates 4d, terminating reaction, by zymotic fluid suction filtration, extraction
Take, reclaim, the ginseng extraction powder after being fermented；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C is carried out at degreasing
Reason；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh ox
Milk is well mixed, and stirs 12.5min, and being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
The homogenate that step (6) is obtained is taken, the working stock culture in formula is added, ferments 4h under the conditions of being placed in 40 DEG C, it is seen that
The upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root ginseng hair
Ferment Yoghourt, refrigerates under the conditions of being subsequently placed in 4 DEG C.
Embodiment 3
Obtained by the following steps：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 12%, ginseng 16%, working stock culture 10%, surplus is fresh
Milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 2:1；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of the mesh of granularity 80 is ground into；Ginseng is taken, the Ginseng Root Powder of the mesh of granularity 80 is ground into；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 5 times of entrainers are added, the entrainer is 70% ethanol solution, is placed in supercritical extracting equipment
In, 12h is soaked at room temperature, extraction time is 5h, and number of times is 4 times, and temperature is 50 DEG C, and pressure is 40MPa, CO2Flow velocity keeps
12L/h is constant, collects product in separating still and obtains balloonflower root extraction powder；Ginseng Root Powder is taken, 5 times of entrainers are added, overcritical extraction is placed in
In taking equipment, 24h is soaked at room temperature, extraction time is 5h, and number of times is 4 times, and temperature is 50 DEG C, and pressure is 40MPa, CO2Flow velocity
Keep 15L/h constant, collect product in separating still and obtain ginseng extraction powder；；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, beta-glucosidase used
Enzyme, performs Q/NXM010 standards, meets GB25594-2009《National food safety standard food industry enzyme preparation》Standard, by
Suzhou Kunlan Biotechnology Co., Ltd. is provided, and ginseng is extracted into powder, carries out sterilization treatment, adds 3 times of amount pure water, access β-
Glucuroide, is put into constant incubator, pH value 5.0, temperature 60 C, cultivates 5d, terminating reaction, by zymotic fluid suction filtration, extraction
Take, reclaim, the ginseng extraction powder after being fermented；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C is carried out at degreasing
Reason；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh ox
Milk is well mixed, and stirs 15min, and being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
The homogenate that step (6) is obtained is taken, the working stock culture in formula is added, ferments 5h under the conditions of being placed in 45 DEG C, it is seen that
The upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root ginseng hair
Ferment Yoghourt, refrigerates under the conditions of being subsequently placed in 5 DEG C.
Rare ginsenoside assay in the present invention
1st, the preparation of reference substance solution
Ginsenoside Rg1、Rf、Rh1(S)、Rb1、Rc、F1、Rb2、Rb3、F2Reference substance, purchased from Chemical College, Jilin Univ.,
And check qualified；Ginsenoside Re, Rg2、Rd、20(S)-Rg3、20(R)-Rg3、Rh2, PPT, PPD reference substance, self-control and checks
It is qualified；Oleanolic acid, ginsenoside Ro's reference substance, purchased from Nat'l Pharmaceutical ＆ Biological Products Control Institute, take reference substance respectively, use methyl alcohol
Dissolve and constant volume, be made into 19 kinds of ginsenoside reference substance solutions；
2nd, the preparation of need testing solution
Learnt from else's experience balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation, it is 6 batches, accurately weighed, be designated as A1, A2, A3, A4, A5,
A6, takes without the balloonflower root ginseng Yoghourt of beta-glucosidase enzyme fermentation, i.e., extract powder through the balloonflower root that supercritical fluid extraction is obtained
Powder is extracted with ginseng, without microorganism conversion, carries out mixing sterilized homogeneous, inoculation, fermentation with treated fresh milk, obtained
6 batches of samples, are designated as B1, B2, B3, B4, B5, B6；
Each sample, adds water to cook twice, each 2h, merging filtrate respectively, crosses D101 type large pore resin absorption columns
(2.9cm × 20cm), loading speed 2mL/min, adsorbs 4h after loading, wash with water to colourless, then with 80% ethanol elution extremely
Without saponin(e reaction, 80% ethanol eluate is collected, recycling design is evaporated near dry, and residue adds methyl alcohol to dissolve and is transferred to 10mL
In volumetric flask, plus methanol dilution is to scale, shakes up, and is filtered with 0.45 μm of syringe filter, takes subsequent filtrate, obtains final product test sample；
3rd, the selection of chromatographic condition
The type high performance liquid chromatographs (Waters, US) of Waters 1525；The Diode Array Detectors of Waters 2998
Device (Waters, US)；Chromatographic column：Unitary C18(4.6mm×250mm,5μm)；Column temperature：30℃；Detection wavelength
203nm；Flow velocity：1.3mL/min；Mobile phase：Acetonitrile (A) -0.05% phosphate aqueous solution (B) gradient elution, gradient elution program
It is as follows：0~14min (A：20 → 21%), 14~24min (21 → 22%), 24~36min (22 → 23%), 36~52min
(23 → 25%), 55~78min (28 → 30%), 78~96min (30 → 35%), 96~112min (35 → 60%), 112~
127min (60 → 90%), 127~134min (90 → 100%), 134~138min (100%), all components are in 135min
Interior appearance, under this chromatographic condition, saponin component reaches baseline separation with other compositions chromatographic peak, obtains Rd linear regression sides
Journey：Y=189484x -13213 (r=0.9996), PPD equations of linear regression：Y=445875x+32102 (r=0.9967),
Rh2Equation of linear regression：Y=282995x-2520.4 (r=0.9997).19 kinds of ginsenoside reference substance HPLC spectrograms are shown in Fig. 1；
4th, rare ginsenoside assay and interpretation of result
Precision draws the μ L of need testing solution 15, injects liquid chromatograph, test sample HPLC chromatogram is obtained, through beta-glucosidase
The HPLC spectrograms of the balloonflower root ginseng Yoghourt after enzyme fermentation are shown in Fig. 2, without the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation
HPLC spectrograms are shown in Fig. 3.The peak area of ginsenoside chromatographic peak compares monomer in the range of linearity of standard curve in test sample
Ginsenoside peak area-content equation of linear regression, calculates in balloonflower root ginseng yoghurt example 3 kinds of monomer ginsenosides (unit)
Content, the results are shown in Table 1.It is bright through the total saponin content in the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation from result
Aobvious is higher than the total saponin content without beta-glucosidase enzyme fermentation.Wherein, the content of Rd is about unfermentable 20 times, and PPD's contains
About unfermentable 30 times of amount, Rh2Content be about unfermentable 9 times, the content of Rg3 is about unfermentable 5 times.Thus may be used
See, through beta-glucosidase enzyme fermentation, rare ginsenoside Rd, PPD, Rh in balloonflower root ginseng Yoghourt can be significantly improved2Content.
The content of rare ginsenoside in the balloonflower root ginseng Yoghourt of table 1
Following reference《Health food is checked and assessment technique specification》(2003 editions) further illustrate effect of the invention
Really.Test example 1：Con A induction of lymphocyte transformation experiment (mtt assay)
The experimental animal of following test example, refers both to male Balb/c healthy mices, 50, is randomly divided into 5 groups, every group 10
Only, low middle high dose group (by the Yoghourt 250,500,1000mg/kg after beta-glucosidase enzyme fermentation) is set, a feminine gender is right
According to group, a balloonflower root ginseng Yoghourt without beta-glucosidase enzyme fermentation is denoted as group of not fermenting.Oral balloonflower root people is carried out to mouse
Ginseng Yoghourt, one time a day, continuous 7 days.
It is aseptic to take spleen, splenocyte suspension is prepared, cell concentration is 3 × 106Individual/ml.In adding 24 well culture plates, per hole
1mL, adds 70 μ L Con A liquid, is placed in 5%CO2, 37 DEG C of CO2Cultivated in incubator.Added without calf serum during 68h
The μ L/ holes of RPMI RPMI-1640 0.7mL and MTT solution 50, determine OD after 72h570nm。
As a result：The middle and high dosage group mouse of sample drenches slip value apparently higher than control group phase, and through statistics, difference has statistics
Meaning (＜ 0.05), the results are shown in Table 2.
Table 2.Con A induction of lymphocyte conversion results
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
The mice spleen lymphocytes proliferation ability of induction is remarkably reinforced, and can strengthen the immunity of organisms of mouse.
Test example 2：Antibody-producting cell detection
The sheep red blood cell (SRBC) of mouse peritoneal injection 3% (SRBC), 0.25mL/ is only.Spleen is taken after immune 4d, cell suspension is made,
By in splenocyte suspension addition RPMI1640 nutrient solutions, concentration is adjusted to 4 × 106Individual/mL.Prepare agarose slide.Incubate 1.5h,
Complement is added, 1.5h is incubated, hemolysis plaque number is counted.
As a result：The middle and high dosage group of sample can substantially increase hemolysis plaque number, and with control group ratio, difference is statistically significant
(＜ 0.05), the results are shown in Table 3.
The antibody-producting cell detection result of the test of table 3.
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
Mouse antibodies generation can be obviously promoted, strengthens mouse body immunity.
Test example 3：Half hemolytic value (HC50) measure
Intraperitoneal injection 3%SRBC, 0.25mL/ are only.After immune 5d, centrifuging and taking serum is sequentially added into 5% after dilution
SRBC, complement.Incubate 20min, ice bath terminating reaction.Reset and add Dou Shi reagents in centrifuging and taking, mix, stand 10min, determine
OD540nm, hemolysin amount is with HC50Represent.
As a result：The middle and high dosage group HC of sample50With control group ratio, significant difference, statistically significant (＜ 0.05), as a result
It is shown in Table 4.
The measurement result of the half hemolytic value of table 4.
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
Experiment mice humoral immune function can be significantly improved, strengthens immunity of organisms.
Test example 4：Mouse carbonic clearance is tested
Mouse tail vein injection india ink (0.1mL/10g), 2,10min after injection prepared Chinese ink, respectively from angular vein clump
Take the μ L of blood 25 and be added to 2ml 0.2%Na2CO3In solution, OD is determined600nm.Place after death takes liver and spleen, after filter paper blots blood stains
Weigh, calculate phagocytic index.
As a result：The basic, normal, high dosage group H phagocytic index of sample are higher, with control group ratio, significant difference, with statistics meaning
Adopted (＜ 0.05), the results are shown in Table 5.
The mouse carbonic clearance experimental result of table 5.
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
The phagocytosis function of mouse monokaryon macrophage can be improved, strengthens non-specific immune function, so as to strengthen immunity of organisms.
Test example 5：NK cell activity assays (lactic dehydrogenase enzyme process)
It is aseptic to take spleen, splenocyte suspension is prepared, cell concentration is 2 × 107Individual/ml.Take target cell and effector cell each 100
μ L are placed in 96 well culture plates, and target cell maximum release aperture adds target cell and each 100 μ L of 2.5%Triton；5%CO2Incubator
Middle culture 4h, draws the μ L of HC1 30 that the μ L of supernatant 100 add 1mol/L, determines OD490nm。
As a result：Sample high dose group NK cytoactives and control group ratio, significant difference, with statistical significance (＜
0.05) 6, be the results are shown in Table.
Table 6.NK cell activity assays results
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
NK cells in mice activity can be improved, strengthens immunity of organisms.
Test example 6：Delayed allergy
Sheep red blood cell (SRBC) (SRBC) carries out peritoneal immunity, and only, 4d measures vola pedis thickness, measuring point to 0.25mL/ after being immunized
Hypodermic injection 20%SRBC, 25 μ L/ only, measure left back foot plantar thickness 3 times after 24h, average.
As a result：Sample is high, the mouse delayed allergy of middle dose group induction, compared with control group, significant difference, tool
Statistically significant (P ＜ 0.05), the results are shown in Table 7.
The delayed allergy result of the test of table 7.
Conclusion：By with negative control and contrast of not fermenting group, through the balloonflower root ginseng Yoghourt after beta-glucosidase enzyme fermentation,
Mouse delayed allergy can be strengthened, strengthen cellular immune function, so as to strengthen immunity of organisms.
Claims (4)
1. it is a kind of green balloonflower root fermented ginseng Yoghourt, it is characterised in that obtained by the following steps：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 4~12%, ginseng 8~16%, working stock culture 5~10%,
Surplus fresh milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 1:2~2:1；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of granularity 60-80 mesh is ground into；Ginseng is taken, the Ginseng Root Powder of granularity 80-100 mesh is ground into；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 2~5 times of entrainers are added, is placed in supercritical extracting equipment, 10~12h is soaked at room temperature, during extraction
Between be 3~5h, number of times be 2~4 times, temperature be 30~50 DEG C, pressure be 20~40MPa, CO2Flow velocity keeps 12L/h constant, receives
Product obtains balloonflower root extraction powder in collection separating still；Ginseng Root Powder is taken, 2~5 times of entrainers are added, is placed in supercritical extracting equipment,
At room temperature soak 20~24h, extraction time be 3~5h, number of times be 2~4 times, temperature be 30~50 DEG C, pressure be 20~
40MPa, CO2Flow velocity keeps 15L/h constant, collects product in separating still and obtains ginseng extraction powder；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, and beta-glucosidase used is held
Row Q/NXM010 standards, meet GB25594-2009《National food safety standard food industry enzyme preparation》Standard, by ginseng
Extraction powder, carries out sterilization treatment, adds 1~3 times of amount pure water, accesses beta-glucosidase, is put into constant incubator, pH value
4.0~5.0,3~5d is cultivated in temperature 50 C~60 DEG C, and terminating reaction by zymotic fluid suction filtration, extraction, is reclaimed, after being fermented
Ginseng extraction powder；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C carries out ungrease treatment；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh milk is mixed
Close uniform, stir 10~15min, being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
The homogenate that step (6) is obtained is taken, the working stock culture in formula is added, ferments 3~5h under the conditions of being placed in 35~45 DEG C,
Seeing the upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root people
Ginseng fermented yoghourt, refrigerates under the conditions of being subsequently placed in 2-5 DEG C.
2. it is according to claim 1 it is a kind of green balloonflower root fermented ginseng Yoghourt, it is characterised in that：In the step (3)
Entrainer is 70% ethanol solution.
3. the production method of the balloonflower root fermented ginseng Yoghourt of a kind of green as claimed in claim 1, it is characterised in that including under
Row step：
(1), balloonflower root fermented ginseng Yoghurt formulation
1000kg Yoghourts are prepared, raw material accounts for weight ratio and is：Balloonflower root 4~12%, ginseng 8~16%, working stock culture 5~10%,
Surplus fresh milk, the working stock culture is streptococcus thermophilus and lactobacillus bulgaricus, and both ratios are 1:2~2:1；
(2), pretreatment of raw material
Balloonflower root is taken, the Platycodon Root of granularity 60-80 mesh is ground into；Ginseng is taken, the Ginseng Root Powder of granularity 80-100 mesh is ground into；
(3) supercritical fluid extraction of balloonflower root and ginseng effective component
Platycodon Root is taken, 2~5 times of entrainers are added, is placed in supercritical extracting equipment, 10~12h is soaked at room temperature, during extraction
Between be 3~5h, number of times be 2~4 times, temperature be 30~50 DEG C, pressure be 20~40MPa, CO2Flow velocity keeps 12L/h constant, receives
Product obtains balloonflower root extraction powder in collection separating still；Ginseng Root Powder is taken, 2~5 times of entrainers are added, is placed in supercritical extracting equipment,
At room temperature soak 20~24h, extraction time be 3~5h, number of times be 2~4 times, temperature be 30~50 DEG C, pressure be 20~
40MPa, CO2Flow velocity keeps 15L/h constant, collects product in separating still and obtains ginseng extraction powder；
(4) ginseng extracts the microorganism conversion of powder
Using beta-glucosidase, the ginseng extraction powder obtained to step (3) ferments, and beta-glucosidase used is held
Row Q/NXM010 standards, meet GB25594-2009《National food safety standard food industry enzyme preparation》Standard, by ginseng
Extraction powder, carries out sterilization treatment, adds 1~3 times of amount pure water, accesses beta-glucosidase, is put into constant incubator, pH value
4.0~5.0,3~5d is cultivated in temperature 50 C~60 DEG C, and terminating reaction by zymotic fluid suction filtration, extraction, is reclaimed, after being fermented
Ginseng extraction powder；
(5) fresh milk treatment
By fresh milk after checking and accepting and coagulability test is qualified, impurity is filtered to remove, being preheating to 40 DEG C carries out ungrease treatment；
(6) mixing homogeneous is sterilized
The balloonflower root that step (3) is obtained extracts the ginseng extraction powder after powder, the fermentation that step (4) is obtained, and fresh milk is mixed
Close uniform, stir 10~15min, being preheating to 60 DEG C carries out homogeneous, carries out high-temperature short-time sterilization；
(7) it is inoculated with, ferments
The homogenate that step (6) is obtained is taken, the working stock culture in formula is added, ferments 3~5h under the conditions of being placed in 35~45 DEG C,
Seeing the upper strata of breast has a small amount of whey to separate out, and is then quickly cooled to 10 DEG C, and keeps 12h, obtains pure plant balloonflower root people
Ginseng fermented yoghourt, refrigerates under the conditions of being subsequently placed in 2-5 DEG C.
4. it is according to claim 3 it is a kind of green balloonflower root fermented ginseng Yoghourt production method, it is characterised in that：It is described
Entrainer is 70% ethanol solution in step (3).
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