CN105768001A - Bee pollen containing probiotics and preparation method thereof - Google Patents
Bee pollen containing probiotics and preparation method thereof Download PDFInfo
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- CN105768001A CN105768001A CN201610178529.1A CN201610178529A CN105768001A CN 105768001 A CN105768001 A CN 105768001A CN 201610178529 A CN201610178529 A CN 201610178529A CN 105768001 A CN105768001 A CN 105768001A
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- bee pollen
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2400/00—Lactic or propionic acid bacteria
- A23V2400/11—Lactobacillus
- A23V2400/169—Plantarum
Landscapes
- Jellies, Jams, And Syrups (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The present invention discloses bee pollen containing probiotics and a preparation method thereof. Bee pollen is used as a main raw material, and scientifically combined with natural plant resource enzymes high in enzyme activities and rich in enzymes, and pupae and larvae intestinal extract containing the most original and the most comprehensive enzymes in the formation of the bee pollen of queen bee larvae and male bee pupae saliva, and intestinal enzymes. A microwave high-temperature instantaneous puffing technology and a probiotic fermentation technology are used. A special biological enzyme digestion, a microwave high-temperature instantaneous low-temperature puffing technology and the probiotic fermentation are used to conduct a rapid, effective and deep wall breaking for the bee pollen to prepare wall broken bee pollen. Then the wall broken bee pollen powder is scientifically combined with one or various functional accessory materials to prepare the health-care bee pollen containing probiotics. The bee pollen powder maximally maintains natural flavors and nutrients, and is high in wall broken rate, high in efficiency, and low in costs.
Description
Technical field
The present invention relates to bee pollen, be specifically related to a kind of bee pollen containing probiotic bacteria and preparation method thereof.
Background technology
The pollen load that bee pollen is taken back when referring to honeybee producting honey, the pollen formed after storage and fermentation in Nidus Vespae.Apis is when gathering honey, and corbiculate leg can collect pollen, forms pollen load, and after entering Nidus Vespae, pollen load can be stored.Its main dietetic therapy composition is: protein, aminoacid, vitamin, bee pollen element, trace element, organized enzyme, flavone compound, lipid, nucleic acid, brassin, phytic acid etc..Wherein amino acid content and ratio are closest to the amino acid pattern that FAO (Food and Agriculture Organization of the United Nation) (FAO) is recommended, and this is extremely rare in wholefood.Bee pollen is the concentrate that the nutritious material being worth with drug effect forms, and it is containing protein, carbohydrate, mineral, vitamin and other active substance.Bee pollen is fabulous natural nutrient food, is also a kind of desirably tonic simultaneously, and has certain medical function.
Bee pollen derives from the Nature, the pollen grain that to be Apis gather in phanerogam (nectariferous plant and plant of pollen) pistil, and add special glandular secretion thing (nectar and saliva) and mixed a kind of irregular discoid shape thing.Bee pollen has natural health effect and medical treatment and its cosmetic values of uniqueness, recognized by increasing people, it it is a kind of high protein and low fat nutritional health food, it is described as " all-round nutraceutical " " the natural Drug Storage of concentration " " cosmetics for oral administration " etc., is the rarity in mankind's wholefood.
Compared with other natural product, the structure comparison of pollen is special.Pollen grain is to be made up of pollen wall, germinal aperature and protoplasm three part.Pollen wall, also known as sporoderm, can be divided into inner and outer wall, and inwall is innermost one layer of sporoderm, smooth, thin and flexible.Outer wall is thicker, hard and lack flexibility, be that in sporoderm, structure is more complicated one layer, and main component is sporopollenin, and this material has a resistance effect of physics, chemistry and enzyme, acidproof, alkaline-resisting, heatproof, pressure, and gastric acid and other digestive system enzyme is also highly stable.Research proves: the nutrient substance in non-pollen (processed by breaking wall) is not easily digested, and the active substance that breaking cellular wall can make pollen fully discharges, so that effect of its pollen product and health care all increase substantially.Pollen wall both have impact on absorbing of pollens nutrition composition, also prevents the release of nutrient substance during extraction, greatly limit the deep development of pollen and utilization, and therefore, the breaking cellular wall of bee pollen is critically important.
The method of pollen broken wall is broadly divided into mechanical breaking-wall method, physical wall breaking and biological wall breaking both at home and abroad at present.
Mechanical breaking-wall method mainly relies on the effects such as the extruding of machinery, shearing, makes pollen wall and interior membrane vesicle break, and the content release of pollen, common have whirlwind comminuting method and micronizing method.Hao Xiaoliang etc. adopt whirlwind comminuting method to have studied the breaking cellular wall of Pollen Pini, and Cao Longkui etc. adopts the ultrafine powder technical research breaking cellular wall of Pollen Maydis, makes pollen particles particle diameter reach less than 8 μm, and epigranular is reasonable.The preparation method that Chinese patent CN103181511 discloses a kind of ultramicro safflower bee pollen, it is characterized in that safflower bee pollen is in the freezer of temperature-20～-30 DEG C after freezing 5～8 days, move on in the calorstat or greenhouse that temperature is 65～75 DEG C and place 20～30 minutes, moving on to temperature again is that 45～50 DEG C of drying baker dry 3～5 hours, move on to water-cooled after dry cooling and grind pulverizing in super micron mill, coolant outlet water temperature controls below 25 DEG C, smashing fineness controls at 1500～2000 orders, after the ultramicro safflower bee pollen sterilization ground, pack by foil sealing, it is housed in temperature for stand-by in-5～7 DEG C of freezers.Use the super-micro wall-broken safflower bee pollen prepared of the inventive method, it is possible to improve the absorbance of safflower bee pollen active ingredient, give full play to safflower bee pollen to cardiovascular and cerebrovascular disease, improve blood circulation and have prevention and effect for the treatment of.Chinese patent CN101449754B discloses the production method of a kind of broker wall bee pollen granules, belongs to the processing technique of bee pollen.The sporoderm-broken rate that current existing broken wall of melissa pollen technology has is low, and some equipment is complicated, and some breaking cellular wall speed is slow etc..The present invention provides a kind of breaking cellular wall efficiency high, sterilization effect is good, the simple broker wall bee pollen granules production method of use equipment, including sterilizing, breaking cellular wall, pelletize and inspection process, it is characterized in that: described wall-breaking machine is breaking cellular wall by vibrating and grinding machine, and the frequency of vibration of this breaking cellular wall by vibrating and grinding machine is between 1200-1600cpm, and Oscillation Amplitude is between 3.5-5mm, every batch of inventory of described bee pollen is 100kg, and broken time is 2 hours.But, extruding that mechanical breaking-wall method sporoderm-broken rate energy consumption is high and strong, shear the loss being likely to result in nutritional labeling.
Physical wall breaking is made by the physical actions such as radiation, swelling, infiltration, ultrasound wave, microwave, difference variation makes pollen wall break, and mainly has temperature differential method, radiation method, hydration breaking cellular wall method, supercritical ultrasonics technology, osmotic pressure breaking cellular wall method, ultralow temperature to add microwave frequency measurment method and liquid nitrogen quenching breaking cellular wall method.Chinese patent CN102114058B is for treating the production method of the bee pollen tablet of fatty liver, relate to product tablet production technology, the bee pollen of remove impurity is immersed in normal pressure and temperature water after comminution by gas stream broken wall treatment, after ultrasonic Treatment, through the refrigerated centrifuger centrifugal treating that rotating speed is 8000～10000 turns, take supernatant, then by supernatant under the ambient temperature of-5～5 DEG C, cross the molecular sieve of below 2800D, isolate the molecular weight bee pollen hydrotrope less than 2800D；After the molecular weight chilled vacuum drying of the bee pollen hydrotrope less than 2800D, obtain the water content Herba leucadis ciliatae lyophilized powder lower than 4%；By described Herba leucadis ciliatae lyophilized powder and water soluble starch mixing film-making.Chinese patent CN101416763B discloses a kind of bee pollen beverage and preparation method thereof, and including bee pollen is carried out pretreatment, broken wall treatment method that breaking cellular wall by temperature difference combines with mechanical breaking-wall method, cold preservation stand, are centrifuged, filtration, constant volume, the multiple working procedure such as fill prepare the Herba leucadis ciliatae powder of clarification.But, single physical method sporoderm-broken rate is relatively low and some equipment requirements is high.
Biological wall breaking is because sporoderm-broken rate is high, shell-broken effect is notable；Action condition is gentle, and heat-sensitive nutrition destructiveness is little；May additionally facilitate bee pollen digestion while breaking cellular wall and absorb, producing the plurality of advantages such as functional metabolic product and be widely used in technology for broken wall of melissa pollen and relevant bee pollen product, the fermentation that biological wall breaking is big mainly includes breaking wall by fermentation and enzymolysis breaking cellular wall.
Enzymolysis breaking cellular wall is to utilize enzyme to make some ingredient breakdown on pollen wall destroy pollen wall, makes germinal aperature open, and pollen content flows out.Enzyme conventional in current enzymolysis breaking cellular wall has cellulase, hemicellulase, pectase, protease, amylase, compound enzyme etc..Chinese patent CN102746413B discloses a kind of bee pollen polysaccharide enzymolysis breaking cellular wall in conjunction with the ultrasonic leach extraction method of hot water, comprises the following steps: dried by bee pollen, remove impurity, pulverize, sieve；Enzymolysis breaking cellular wall；Heat ultrasonic lixiviate；Dehydration；Perchloric acid removing protein；DEAE column chromatography, eluting；CTAB separates containing acidic polysaccharose precipitation and precipitates containing neutral polysaccharide；Dissolve polysaccharide deep after through dehydration, vacuum freezing, dry obtain the pure bee pollen polysaccharide of high-purity.Chinese patent CN101401821B discloses one and through protease hydrolyzed, bee pollen is become polypeptide, tablet made by polypeptide and preparation method thereof.It is will pulverize through screening, roguing, dried bee pollen, reaches requirement post-treatment skill water, stir, be positioned over less than-18 DEG C freezing 24-48 hour.The fresh water (FW) stirring adding 100 DEG C after taking-up immediately is dissolved, and with homogenizer homogenizing 20-60 minute, puts into and adds cellulase in enzymatic vessel and carry out enzymolysis 0.5-2 hour, after adjust pH value with alkali liquor after add protease and carry out enzymolysis 3-6 hour.Immediately bee pollen peptide liquid is heated after enzymolysis and carry out enzyme denaturing.With starch for excipient, carry out pelletize, tabletting and get final product.It is characterized as being 1) specificity: protease, pectase, cellulase, amylase can single-minded pollen enzymolysis wall and the protein at germinal aperature place, pectin, cellulose, starch grain macromolecular substances；2) high efficiency: enzyme digestion reaction shell-broken effect is good；3) reaction condition is gentle, is conducive to the preservation of nutrient substance；But due to the single-minded characteristic of bee pollen sporoderm complicated component and enzyme, existing enzyme preparation and composite, it is impossible to well solving enzymolysis breaking cellular wall problem, simultaneously as yielding poorly of enzyme, expensive, enzymolysis cost is high.
Breaking wall by fermentation method is mainly by the various enzymes produced in the probiotics sweats such as yeast, aspergillosis, lactic acid bacteria, and the various enzymes contained inside pollen, the passage portion (germinal aperature, ditch) of pollen inside and outside wall is got through in the effect utilizing these enzymes, so that the nutritional labeling dissolution in inwall.Strain conventional in current breaking wall by fermentation has aspergillosis, yeast, lactic acid bacteria, Pleurotus ostreatus, Lentinus Edodes etc..Chinese patent CN101756091B discloses a kind of pollen natto tablet, including the component of following weight portion: pollen 4～5 parts, and natto 3～4 parts, 1～2 part of Monas cuspurpureus Went, oligosaccharide 1～2 part.Described pollen is with bee pollen for raw material, obtains through following preparation method: first with 50～100PPMClO2Aqueous solution sprays the airtight sterilization of raw material bee pollen 30 minutes, and then adjusting humidity is 25%, and inoculating starter ferments, temperature 30～37 DEG C, ferments 12 hours, checks acidity, as pH=4.5, and cold drying, obtain pollen；Described leaven is streptococcus acidi lactici and lactobacillus.The fermentation process of Chinese patent CN104286623A bee pollen and bee pollen, adopts probiotic bacteria mixed solid fermentation bee pollen, and production cost is low, and the fund of investment is less, convenient in downstream, pollutes little.The fermentation bee pollen produced, it is possible to regulate human body intestinal canal function.The protein contained in bee pollen is broken down into little peptide and free amino acid, is conducive to digesting and assimilating of the intestines and stomach, removes anaphylactogen.Described probiotic bacteria adopts Bifidobacterium lactis or Lactobacillus plantarum or bacillus acidophilus.The viable count of described probiotic bacteria is 1 × 109-5×109CFU/g.The manufacture method of a Chinese patent CN104059842A bee pollen wine, the step of the manufacture method of described bee pollen wine is as follows: takes the bee pollen 0.8-1 weight portion that sterilization treatment crosses and is cooled to 30-35 DEG C, add the cold boiled water of softening that the black clothing song of 1.5 2 weight portions adds 0.8--1 weight portion, put into the stirring of dispensing basin all, put into sealing and fermenting 40 48 hours in the wine vat that sterilization treatment is crossed；Add the cold boiled water of softening of 10 15 weight portions, add the Mel of 10 15 weight portion 40 41 concentration, Arillus Longan core 1.5 2.5 weight portion that addition processed, it is separately added in feed liquid, being stirred uniformly, blanking is complete to be sealed, temperature during regulation and control fermentation, being maintained between temperature 20 25 DEG C, the wine vat that falls is 30 35 days to fermentation dwell time；Extracting the wine liquid after filtering, water proof heat sterilization temperature 85 90 DEG C, 20 30 minutes, seal precipitation up for safekeeping 15 20 days after cooling, fill finished product can be waited to drink.Chinese patent CN102389069A discloses a kind of broken wall of melissa pollen method, it is characterised in that comprises the steps: addition culture propagation in bee pollen, makes broken wall of melissa pollen；Yeast affiliated in bee pollen is calculated as the 0.5～3% of bee pollen weight with dry weight；After bee pollen adds yeast, add the water of bee pollen volume 1.5～2.5 times, then ferment 2～4 days in 28～34 DEG C, finally heat 4～8h in 36～38 DEG C, obtain the bee pollen solution after breaking cellular wall.Chinese patent CN103598654B discloses a kind of pollen active probiotic drink and preparation method thereof.This pollen active probiotic drink is with bee pollen for raw material, is equipped with a certain proportion of maltose and/or galactose, through lactobacillus BL1 and two kinds of probiotic mixed fermentations of lactobacillus BL2, forms then through allotment.The present invention is directed to bee pollen raw material acidity relatively strong, the feature not easily utilized by microorganism, it is preferable that two acid resistances are strong and have the bacterial strain of multiple merit, make number of live bacteria of probiotics in product 30 days be maintained at 10 by controlling fermentation technology11More than cfu/L, and a large amount of flavone aglycone can be obtained by bioconversion bee pollen flavonoid glycoside, improve the bioavailability of bee pollen flavone.Simultaneously rich in other bee pollen and probiotics fermention product, nutritious, aromatic flavor, sour and sweet palatability, there is the functions such as looks improving and the skin nourishing, enhancing immunity, reduction cholesterol, regulating intestinal canal flora and Constipation simultaneously.Aspergillosis fermentation method weak flavor in above-mentioned breaking wall by fermentation method；When oyster mushroom fermentation method and the inoculation of mushroom ferment method, easy miscellaneous bacteria infects, mycelial growth is slow, antibacterial ability is poor, sporoderm-broken rate is low；Fermented by lactic acid bacteria and yeast fermentation method sporoderm-broken rate are high, nutrient component damages is little, amino acid content is high but time length, speed are slow after breaking cellular wall.
To sum up, seeking one and keep bee pollen natural flavour mountaineous to greatest extent and nutritional labeling, the broken wall of melissa pollen method that sporoderm-broken rate is high, efficiency is high, cost is low, the field of deep to expand bee pollen is necessary.
Summary of the invention
Solved by the invention technical problem is that the defect overcoming existing broken wall of melissa pollen technology, with bee pollen for primary raw material, enzyme activity is high, natural plant enzyme that enzyme system is abundant and the most original containing bee pollen forming process, the pupa worm intestinal extract science of queen bee nit that enzyme system is the most complete and drone pupa saliva and intestinal enzyme system is composite, and adopt the instantaneous puffing technique of microwave high-temperature and probiotics fermention technology, by Specialty bio enzymolysis, bee pollen is carried out quickly by the instantaneous low temperature expanding of microwave high-temperature and probiotics fermention, effectively and degree of depth breaking cellular wall, prepare broker wall bee pollen, then composite with one or more functional auxiliary material science, prepared one keeps bee pollen natural flavour mountaineous and nutritional labeling to greatest extent, sporoderm-broken rate is high, efficiency is high, cost is low, health care bee pollen containing probiotic bacteria.
In order to achieve the above object, the present invention is by the following technical solutions:
A kind of bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 150-180 part, plant extract 10-30 part, pupa worm intestinal extract 20-30 part, modified dietary fiber 20-30 part, oligosaccharide 7-15 part, Lactobacillus plantarum powder 6-10 part, hydroxyl isomaltulose 2-6 part, mango powder 2-6 part；
Preferably, the described bee pollen containing probiotic bacteria, mainly have the raw material of following parts by weight to prepare:
Bee pollen 160-170 part, plant extract 15-25 part, pupa worm intestinal extract 23-27 part, modified dietary fiber 23-27 part, oligosaccharide 9-13 part, Lactobacillus plantarum powder 7-9 part, hydroxyl isomaltulose 3-5 part, mango powder 3-5 part；
It is highly preferred that the described bee pollen containing probiotic bacteria, the raw material of following parts by weight is mainly had to prepare:
Bee pollen 165 parts, plant extract 20 parts, pupa worm intestinal extract 25 parts, modified dietary fiber 25 parts, oligosaccharide 11 parts, 8 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 4 parts, mango powder 4 parts；
Further, the described bee pollen containing probiotic bacteria also includes one or more adjuvants of following parts by weight；
Algae 0.6-1.2 part, corn oligopeptide powder 0.5-1 part, pueraria root powder 0.5-1 part, fish oil 0.2-0.8 part, plant sterol 0.2-0.8 part, phosphatidyl serine 0.1-0.7 part, oligochitosan 0.1-0.7 part, marine fishbone collagen oligopeptide powder 0.1-0.7 part, Chinese herbal medicine 0.1-0.6 part, Cordyceps militaris (L.) Link. 0.1-0.5 part；Soybean protein isolate 0.1-0.5 part；
Preferably, the described bee pollen containing probiotic bacteria also includes one or more adjuvants of following parts by weight；
Algae 0.8-1.0 part, corn oligopeptide powder 0.7-0.9 part, pueraria root powder 0.7-0.9 part, fish oil 0.4-0.6 part, plant sterol 0.4-0.6 part, phosphatidyl serine 0.3-0.5 part, oligochitosan 0.3-0.5 part, marine fishbone collagen oligopeptide powder 0.3-0.5 part, Chinese herbal medicine 0.2-0.4 part, Cordyceps militaris (L.) Link. 0.2-0.4 part；Soybean protein isolate 0.2-0.4 part；
It is highly preferred that the described bee pollen containing probiotic bacteria also includes one or more adjuvants of following parts by weight；
0.9 part of algae, corn oligopeptide powder 0.8 part, pueraria root powder 0.8 part, 0.5 part of fish oil, plant sterol 0.5 part, phosphatidyl serine 0.4 part, oligochitosan 0.4 part, marine fishbone collagen oligopeptide powder 0.4 part, Chinese herbal medicine 0.3 part, Cordyceps militaris (L.) Link. 0.3 part；Soybean protein isolate 0.3 part；
Further, described bee pollen is any one in bee pollen of maize, Semen Sesami bee pollen, Brassica campestris L pollen, pollen of Semen Fagopyri Esculenti；
Further, described plant extract does not contain only the various plants enzymes such as abundant plant rennet, amylase, hemicellulase, esterase, oxidoreductase and containing the nutrient substance such as vegetable polysaccharides and monosaccharide, plant amylum, vegetable protein, not only can provide comprehensive, natural phytoenzyme for the bee pollen containing probiotic bacteria, the nutrient substance that also can provide comprehensively for probiotic bacteria, enrich, composite with pupa worm intestinal extract science, sporoderm-broken rate is higher, better effects if；
Preferably, described plant extract adopts the extract at low temperature technology such as ultrasonic cleaning, microwave-assisted supersound extraction and high-pressure pulse electric extraction, concentrating under reduced pressure, is effectively increased raw material availability, phytoenzyme activity and productivity；It is effectively ensured the foodsafety of plant extract；
It is highly preferred that the preparation method of described plant extract is: by Fructus Hordei Germinatus and Fructus Tritici aestivi 8-10:1-3 Homogeneous phase mixing in mass ratio, be crushed to granularity 0.5-1mm, obtain pulverizing Fructus Hordei Germinatus；nullThen by Fructus Chaenomelis、Fructus Ananadis comosi、Fructus Fici in ultrasonic washing unit at power 200W、Ultrasonic cleaning 5-10min when frequency 30KHz，Drain，It is crushed to granularity 0.5-1mm under room temperature，And 7-9:1-3:1-2 Homogeneous phase mixing in mass ratio，The pulverizing Fructus Hordei Germinatus adding mixture quality 3-5 times obtains raw mixture，Add the water of raw mixture quality 1-3 times，It is 3-4 with Fructus Citri Limoniae acid for adjusting pH value，At power 150-300W、Microwave extraction is carried out when frequency 2000Hz，Wherein，Each microwave exposure total time 60-80s,Carry out compartment irradiation: irradiation 10s，Interval 10s，Control temperature 20-35 DEG C，Such irradiation 10 times，Simultaneously at power 200-300W,Ultrasonic assistant extraction is carried out when frequency 30-40KHz；Insulation 1-3h, then, microwave extraction is carried out when power 200-400W, frequency 2000Hz, wherein, each microwave exposure total time 90-105s, carry out compartment irradiation: irradiation 15s, interval 10s, controls temperature 40-60 DEG C, such irradiation 10 times, simultaneously at power 300-500W, carry out ultrasonic assistant extraction when frequency 40-50KHz, be finally naturally cooling to room temperature, in electric field intensity 25-35kV/cm, burst length 400-600 μ s, carries out high-pressure pulse electric and extracts 15-20min when pulse frequency 200-300Hz；Extracting solution filters to obtain the first filtrate, adds the water rinsing of filtering residue 4 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:3-5 Homogeneous phase mixing in mass ratio, being evaporated to solid content is more than 20%, obtains plant extract；
Preferably, in described ultrasonic washing unit, cleanout fluid is the sodium bicarbonate solution of 0.3-0.5%.
Further, described pupa worm intestinal extract is with the queen bee nit intestinal of 2-3 age in days and the drone pupa intestinal of 11-12 age in days for raw material, obtains through freeze proof protection, freezing crushing, biological demulsifying and centrifugation；
nullPreferably，The preparation method of described pupa worm intestinal extract，Comprise the steps: the drone pupa intestinal 3-5:1-3 in mass ratio mixing of the queen bee nit intestinal by 2-3 age in days and 11-12 age in days，Add the sericin peptide taken solution that mass percent is 8-12% of mixture quality 1-2 times，Stir，Put-18-22 DEG C of freezing 5-10min，Broken，Grind to form homogenate，Add the biological demulsifying agent breakdown of emulsion 20-40min of its quality 0.03-0.05%,At 18000-25000g，Centrifugal 20-30min under 4 DEG C of conditions，Collect the middle level liquid after being centrifuged for the first time，Then at 18000-25000g，Centrifugal 20-30min under 4 DEG C of conditions，Collect the middle level liquid after second time is centrifuged，Obtain pupa worm intestinal extract；
Preferably, the pH value of described sericin peptide taken solution is 6-7；
Preferably, described biological demulsifying agent is that glycolipid class, lipopeptid class, cell wall are in conjunction with the one or more combination in class biological demulsifying agent；
Preferably, the quality group of described biological demulsifying agent becomes: glycolipid class: lipopeptid class: cell wall is in conjunction with class=5-7:4-6:2-4；
Preferably, described glycolipid class biological demulsifying agent is one or both combinations in rhamnolipid, alkyl polyglucoside；
It is highly preferred that the quality group of described glycolipid class biological demulsifying agent becomes: rhamnolipid: alkyl polyglucoside=3-5:1-2.
Further, described modified dietary fiber is that through physics, chemical or biological method having of processing and obtain, the abundant soluble fiber cellulose content of strong retentiveness, dilatancy, thickening property, adsorptivity and network is high, biological activity is strong, human body beneficial flora has cellulose important, positive role by dietary fiber, compared with full diet fiber, its biological action is more powerful, also can be greatly prolonged the shelf-life of bee pollen containing probiotic bacteria；
Preferably, described modified fibre is to be obtained through enzyme enzymolysis by one or more in inulin, apple fiber, Herba avenae fatuae fiber, Semen Tritici aestivi fiber；
More preferably, the preparation method of described modified dietary fiber, comprise the following steps: by inulin, Semen Tritici aestivi fiber, Herba avenae fatuae fiber 5-7:2-4:1-3 Homogeneous phase mixing in mass ratio, add its water of quality 3-7 times, room temperature 100-300W, 35-40KHz condition supersound extraction 10-15min, then at electric field intensity 20-40kV/cm, burst length 300-500 μ s, carry out high voltage pulse electric field processing 10-15min when pulse frequency 200-400Hz；It is 4.5-6.5 with breast acid for adjusting pH value, adds the enzyme of mixture quality 0.1-0.3%, in 45-55 DEG C of enzymolysis 20-48min；Enzymolysis solution filters, and filtrate reduced in volume, lyophilization to moisture are that namely 5-8% obtains modified dietary fiber；
Described enzyme is cellulase, xylanase, laccase, pectase 1-3:1-3:1-2:1-2 Homogeneous phase mixing in mass ratio.
Further, described oligosaccharide is at least one in oligofructose, stachyose, Raffinose, oligomeric xylose, oligomeric galactose, soybean oligo saccharide, oligomeric isomaltose, Oligomeric maltose；
Preferably, described oligosaccharide parts by weight consist of oligofructose 40-50 part, Oligomeric maltose 30-40 part, Raffinose 20-40 part, soybean oligo saccharide 16-18 part, oligomeric galactose 10-15 part, oligomeric xylose 10-15 part, oligomeric isomaltose 10-15 part, stachyose 8-12 part.
Further, described Lactobacillus plantarum powder is prepared with Lactobacillus plantarum CGMCCNO.11763 according to a conventional method for starting strain, and its viable bacteria content is: 7 × 1012-9×1012cfu/g；The cryoprotective agent that wherein freeze drying process adopts is with the animal or plant containing antifreeze protein for raw material; prepared through high-pressure pulse electric extraction, ultrasonic assistant microwave extraction and compound enzyme enzymolysis, Lactobacillus plantarum powder can be effectively improved at freezing dry process Viable detection；
Preferably, described protectant preparation method, comprise the steps: winter rye, Caulis et Folium Ammopiptanthi Mongolici, sharkskin collagen protein is respectively washed, drain, 8-10:3-5:2-4 Homogeneous phase mixing in mass ratio, add the lactic acid moistening 3-8h that pH value is 3.8-4.5 of mixed material quality 0.1-1 times, pulverize immediately after-18--22 DEG C of freezing 1-2h, freezing thickness of feed layer 3-5cm, ground product particle diameter 0.5-3mm, it is subsequently added into the water of ground product quality 10-20 times, it is 3.5-5.5 with breast acid for adjusting pH value, at electric field intensity 25-35kV/cm under room temperature, burst length 300-500 μ s, high voltage pulse electric field processing 20-30min is carried out when pulse frequency 200-300Hz；Then carry out microwave irradiation and extraction 15-20min when power 150-300W in room temperature, simultaneously at power 200-300W, when frequency 30-40KHz, carry out ultrasonic assistant extraction；Add the compound enzyme of extracting solution quality 1-2%, in 45-55 DEG C of enzymolysis 30-50min；Enzymolysis solution filters, filtrate concentrates, low-temperature grinding to particle diameter is that namely 0.1-0.3mm obtains protective agent；
Described compound enzyme is cellulase, protease, amylase, pectase, tannase 2-4:1-3:1-3:1-2:1-2 Homogeneous phase mixing in mass ratio.
Further, described algae is any one in Haematocoocus Pluvialls, salt alga, spirulina plalensis.
Further, described Chinese herbal medicine is to prepare through micronizing with one or more in Flos Chrysanthemi, Flos Sophorae, Radix Ginseng, Bulbus Lilii, Radix Puerariae, Fructus Lycii, Herba Taraxaci, Rhizoma Dioscoreae, Poria, Fructus Hippophae, Flos Rosae Rugosae, Flos Lonicerae, Herba Menthae, Fructus Momordicae, Colla Corii Asini, Fructus Jujubae, Folium Mori, Fructus Momordicae charantiae, Agaricus blazei Murrill.
The preparation method that another object of the present invention is to provide the above-mentioned bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
null2) bee pollen is put in the ultrasonic washing unit equipped with 0.3-0.5% sodium bicarbonate solution，In 200W under room temperature、40KHz cleans 3-5min，Rinsing，Drain，The sericin peptide taken solution that mass percent is 13-18% soaks 5-7min，Take out，Put-18-22 DEG C of freezing 5-10min，Broken，Immediately at power 3-5Kw,Frequency 2450MHz,Temperature 120-140 DEG C of microwave heating 5-7s,Add the water of former bee pollen quality 3-5 times，It is 4-6 with breast acid for adjusting pH value，In electric field intensity 20-40kV/cm，Burst length 400-600 μ s,High voltage pulse electric field processing 10-15min is carried out under pulse frequency 200-400Hz room temperature condition，Then room temperature 400W、40KHz condition supersound process 10-15min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 40-50 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 10-15min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution, add the modified dietary fiber of oligosaccharide, 20-30%, fully dissolve 5-10min, it is naturally cooling to 40-45 DEG C, add the 50-60% ferment at constant temperature 3-5h of Lactobacillus plantarum opaque amount, then with the ramp of 0.8-1.0 DEG C/min to 50-55 DEG C, add the 40-50% ferment at constant temperature 1.5-3.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 40-45 DEG C with the speed of 0.6-0.8 DEG C/min, continue fermentation 0.5-1h, fermentation liquor 100-300 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 25-35min with hydroxyl isomaltulose, mango powder and adjuvant, speed of agitator 20-40r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 12-18min, speed of agitator 40-60r/min, namely sterile filling, sealing, packaging obtain the bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to more than 98%, in the bee pollen containing probiotic bacteria, Lactobacillus plantarum viable count is up to 6 × 1011-9×1011cfu/g。
Lactobacillus plantarum of the present invention (Lactobacillusplantarum) XH is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 30th, 2015, preserving number is CGMCCNO.11763, preservation address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101.
Lactobacillus plantarum probiotic properties is as follows:
Lactobacillus plantarum CGMCCNO.11763 provided by the present invention is found to survive when pH is 1.50 through experiment, still in existing state after 1% cholate is cultivated 4 hours；Lactobacillus plantarum CGMCCNO.11763 degrading nitrite speed is fast, and capacity of decomposition reaches 10.9mg/h/kg, and this strain is when producing Pickles, and whole sweat nitrite concentration is at below 4.8mg/kg；Degrading rate of cholesterol, after fermentation 60h hour, can be reached 64.76% by CGMCCNO.11763.CGMCCNO.11763 Adhering capacity measure from coagulation rate be 95.71%.
Lactobacillus plantarum CGMCCNO.11763 is to cholesterol degradation capability study and mensuration:
Take 1mlCGMCCNO.11763 mother solution and be inoculated in MRS cholesterol fluid medium (the cholesterol level 0.1mg/ml of 10mL, pH6.2) in, the constant temperature of 37 DEG C stands and cultivates 20h respectively, 40h, 60h is standby, with the MRS cholesterol culture medium that accesses 1mL sterilized water for comparison, take bacteria liquid sample and the comparison each 1ml of liquid of above cultivation different time, 9000r/min, centrifugal 10min at 4 DEG C, obtain fermented supernatant fluid, in o-phthalaldehyde method mensuration supernatant, cholesterol level is (particularly as follows: take each supernatant 0.1ml in corresponding test tube, add glacial acetic acid 0.3ml, the o-phthalaldehyde(OPA) 0.15ml of 1mg/ml, it is slowly added into concentrated sulphuric acid 1.0ml, mix homogeneously.Room temperature stands 10min, surveys light absorption value under 550nm).Each process 3 repetition, in kind makes cholesterol standard curve, calculates cholesterol level and degradation rate in supernatant, and result is in Table 1.It can be seen that cholesterol is had good Degradation by CGMCCNO.11763, after fermentation 60h hour, degradation rate can reach 64.76%.
The table 1 degraded situation to cholesterol.
| Degradation time (h) | 0 | 20h | 40h | 60h |
| Cholesterol level (mg/ml) | 0.2273±0.0058 | 0.1356±0.0018 | 0.1011±0.0094 | 0.801±0.0231 |
| Degrading rate of cholesterol % | 40.34% | 55.52% | 64.76% |
The bile tolerance test of Lactobacillus plantarum CGMCCNO.11763 bacterial strain:
Take CGMCCNO.11763 bacterium solution 1mL and inoculate strain in the 10mLMRS fluid medium (PH=6.4) containing different cholate (concentration gradients is 0.0%, 0.2%, 0.4%, 0.6%, 0.8%, 1%), be placed at 37 DEG C to cultivate respectively 0,2,4h, each process 3 repetition.Respectively take 1ml sample bacterium solution to mix in 9ml normal saline, prepare dilution factor solution, take 0.1ml diluent to be coated with in MRS, be inverted in 37 DEG C of biochemical cultivation cases and cultivate 48 hours (each dilution factor do 3 parallel) record and calculate the several number of bacterium on flat board.Result is in Table 2.This bacterium known increment of bacterium after gallbladder salinity is 1% process 4h still reaches 0.59 ± 0.92 × 107(cfu/ml), there is good bile tolerance ability.
Table 2 bile tolerance ability detection [(± s) × 107cfu/ml]
The acid resistance test of Lactobacillus plantarum CGMCCNO.11763 bacterial strain
Take HLX37 mother solution and inoculate strain in the 10mLMRS fluid medium of different pH value (pH gradient is 1.5,2.0,2.5,3.0,3.5,4.0) by 1ml, be placed at 37 DEG C to cultivate respectively 0,2,4h, each process 3 repetition.Respectively take 1ml sample bacterium solution to mix in 9ml normal saline, prepare dilute solution, take 0.1ml diluent and be coated with in MRS, in 37 DEG C of biochemical cultivation cases, be inverted the bacterium colony number cultivated on 48 hours (each dilution factor do 3 parallel) record flat board.Result is in Table 3.Illustrate that this bacterium has very strong acid-fast ability.
Table 3 acid-fast ability detection [(± s) × 107cfu/ml]
The Adhering capacity of Lactobacillus plantarum CGMCCNO.11763 measures
Cultivate CGMCCNO.11763 (MRS fluid medium), bacillus coli DH 5 alpha (LB fluid medium) 24h obtains fermentation liquid, be respectively placed in 3000r/min, centrifugal 10min at 4 DEG C, collect bacterium mud, wash bacterium mud 2 times with the sterile phosphate buffer (PBS) of pH=7.0 respectively and (in bacterium colony, namely add PBS, after concussion mix homogeneously, be placed in 3000r/min, centrifugal 10min at 4 DEG C, collect thalline).From coagulation rate (%): with aseptic PBS, bacterium mud CGMCCNO.11763 to be formed in the suspension bacteria liquid that light absorption value is 0.4 ± 0.1 (A0) and the bacteria suspension at wavelength 600nm place, measure light absorption value A24 after standing 24h, be (A0 A24)/A0 from coagulation rate (%) formula.；His coagulation rate (%): the outstanding bacterium solution of CGMCCNO.11763 and bacillus coli DH 5 alpha is adjusted to the mix suspending bacterium solution that the light absorption value at wavelength 600nm place is 0.6 ± 0.1 (A0).Measuring light absorption value A24 after standing 24H, his coagulation rate (%) formula is (A0 A24)/A0.Measurement result is in Table 5, it is known that CGMCCNO.11763 is 95.71% from coagulation rate, has very strong Adhering capacity.
Table 4 Adhering capacity table
The bacterial strain physiological property of Lactobacillus plantarum CGMCCNO.11763
Described Lactobacillus plantarum (Lactobacillusplantarum) XH is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (being called for short CGMCC) on November 30th, 2015, preserving number is CGMCCNO.11763, preservation address is: North Star West Road 1, Chaoyang District, city of BeiJing, China institute 3, Institute of Microorganism, Academia Sinica, postcode: 100101.
This bacterial strain feature is as follows: examine under a microscope, and this bacterial strain is rod-short, and Gram’s staining is positive, atrichia, does not produce spore；On solid medium, this bacterium bacterium colony is white, and smooth surface is fine and close, and form is circular, and edge is more neat.
Physicochemical characteristics is: catalase (-), gelatin liquefaction (-), indole experiment (+), mobility (-), fermentation gas (-), nitrate reductase (-), fermentation gas (-), product hydrogen sulfide gas (-), pH4.0MRS culture medium grows (+).It is accredited as Lactobacillus plantarum (Lactobacillusplantarum), called after Lactobacillus plantarum (Lactobacillusplantarum) XH through Physiology and biochemistry.
Bacterial strain can at 57 DEG C well-grown, glucose tolerance is 275g/L.
Lactobacillus plantarum of the present invention, by gathering people Li Jianshu, separates in Yoghourt from Xinjiang Uygur fellow-villager family and obtains, acquisition time on June 2nd, 2015.
Lactobacillus plantarum CGMCCNO.11763 of the present invention is found to survive when pH is 1.50 through experiment, still in existing state after 1% cholate is cultivated 4 hours；Lactobacillus plantarum CGMCCNO.11763 degrading nitrite speed is fast, and capacity of decomposition reaches 10.9mg/h/kg, and this strain is when producing Pickles, and whole sweat nitrite concentration is at below 4.8mg/kg；Degrading rate of cholesterol, after fermentation 60h hour, can be reached 64.76% by CGMCCNO.11763.CGMCCNO.11763 Adhering capacity measure from coagulation rate be 95.71%, bacterial strain can at 57 DEG C well-grown, glucose tolerance is 275g/L.
Beneficial effect:
The present invention is with bee pollen for primary raw material, enzyme activity is high, natural plant enzyme that enzyme system is abundant and the most original containing bee pollen forming process, the pupa worm intestinal extract science of queen bee nit that enzyme system is the most complete and drone pupa saliva and intestinal enzyme system is composite, and adopt the instantaneous puffing technique of microwave high-temperature and probiotics fermention technology, by Specialty bio enzymolysis, bee pollen is carried out quickly by the instantaneous low temperature expanding of microwave high-temperature and probiotics fermention, effectively and degree of depth breaking cellular wall, prepare broker wall bee pollen, then composite with one or more functional auxiliary material science, prepared one keeps bee pollen natural flavour mountaineous and nutritional labeling to greatest extent, sporoderm-broken rate is high, efficiency is high, cost is low, health care bee pollen containing probiotic bacteria.Concrete test effect is shown in embodiment seven to ten two, and concrete component know-why is as follows:
1. bee pollen, with bee pollen for raw material, is carried out parasite killing initially with ultrasonic cleaning and goes out ovum, killing microorganisms, removal pesticide residues and heavy metal ion etc. by the broker wall bee pollen that prepared by the present invention, substantially increases the foodsafety of bee pollen containing probiotic bacteria；Aqueous solution containing sericin peptide taken soaks, the loss of bee pollen activity material that is chilled and that cause can be reduced to greatest extent, improve the extraction ratio of bee pollen effective ingredient, also establish solid foundation for follow-up alternating temperature probiotics fermention simultaneously；Adopt that microwave high-temperature is instantaneous expanded makes chilled bee pollen appropriateness expanded, enhance between the inside and outside wall of bee pollen and inclusions and the separation, fluffy of the structure of matter own, promote the formation of netted result, enhance the permeability of inside and outside mass exchange, follow-up enzyme molecule, lactic acid bacteria infiltrate into inside and play a role, and the maximization to reach bee pollen effective ingredient accumulates；The extract at low temperature technology such as high-pressure pulse electric, ultrasonic, biological enzymolysis are organically combined, functional materials and the effective ingredient of bee pollen can be retained to greatest extent；The animal ferment source science phytoenzyme high containing multiple enzyme activity, enzyme system is abundant and bee pollen formed is composite, can effectively degrade various macromolecular substances in bee pollen, its abundant enzyme system is that commercially available compound enzyme is incomparable, more meet the original natural rule of " the taking from nature, be used for nature " of the formation of bee pollen material and degraded；With functional plants lactobacillus powder for leaven, adopt temperature-variable fermentation and gradation vaccination ways can obtain the maximum accumulation of proliferation of probiotics and functional metabolic product while bee pollen is carried out further degree of depth breaking cellular wall to greatest extent.Adopt broker wall bee pollen sporoderm-broken rate prepared by said method up to more than 98%.
2. the queen bee nit intestinal of the pupa worm composite 2-3 age in days of intestinal extract science and the drone pupa intestinal of 11-12 age in days that prepared by the present invention are raw material, aqueous solution containing sericin peptide taken soaks, the loss of pupa worm active substance that is chilled and that cause can be reduced to greatest extent, improve the extraction ratio of pupa worm effective ingredient；Use biological demulsifying agent breakdown of emulsion, separating of the hydrolyzed solution that can promote the miscible materials such as the fat in pupa worm intestinal and contain abundant intestinal enzyme, the maximization that can obtain intestinal enzyme is extracted, relative to existing direct centrifugation or the addition separation method such as buffer, biochemicals, intestinal enzyme extraction rate is high, technique is simple, foodsafety is high, environmentally friendly.Meanwhile, pupa worm intestinal extract is through follow-up enzymolysis, possibly together with queen bee nit polypeptide and drone pupa polypeptide, adds more functional bioactive material for the bee pollen containing probiotic bacteria.
3. the Lactobacillus plantarum CGMCCNO.11763 of the present invention is found to survive when pH is 1.50 through experiment, still in existing state after 1% cholate is cultivated 4 hours；Lactobacillus plantarum CGMCCNO.11763 degrading nitrite speed is fast, and capacity of decomposition reaches 10.9mg/h/kg, and this strain is when producing Pickles, and whole sweat nitrite concentration is at below 4.8mg/kg；Degrading rate of cholesterol, after fermentation 60h hour, can be reached 64.76% by CGMCCNO.11763.CGMCCNO.11763 Adhering capacity measure from coagulation rate be 95.71%.
4. the cryoprotective agent that the present invention adopts when preparing Lactobacillus plantarum powder is by composite to the winter rye higher containing antifreeze protein, Caulis et Folium Ammopiptanthi Mongolici and sharkskin collagen protein science; prepare through high-pressure pulse electric extraction, ultrasonic assistant microwave extraction and biological enzymolysis; omnidistance extract at low temperature; antifreeze protein extraction ratio is high; loss is few; the protective agent antifreeze peptide content obtained is high, kind is complete, functional by force, cryoprotective effects is good, improves the viable bacteria content in Lactobacillus plantarum powder.
5. the plant extract that prepared by the present invention does not contain only the various plants enzymes such as abundant plant rennet, amylase, hemicellulase, esterase, oxidoreductase and containing the nutrient substance such as vegetable polysaccharides and monosaccharide, plant amylum, vegetable protein, soluble fiber, not only can provide comprehensive, natural phytoenzyme for the bee pollen containing probiotic bacteria, also can provide comprehensively for Lactobacillus plantarum, abundant nutrient substance；After the extract at low temperature such as ultrasonic cleaning, microwave-assisted supersound extraction and high-pressure pulse electric extraction, concentrating under reduced pressure, its effect is more significantly, and is effectively increased raw material availability, phytoenzyme activity and productivity；It is effectively ensured the foodsafety of plant extract.
6. supersound extraction, high-pressure pulse electric are extracted and biological enzymolysis combination of sciences by the modified dietary fiber that prepared by the present invention, gained modified dietary fiber retentiveness, dilatancy, thickening property be higher and acid and alkali, alkali, salt impact, soluble fiber cellulose content is high, it is easier to be utilized by lactic acid bacteria, improve lactic acid bacteria in the growth of human body intestinal canal and fertility, increase kind and the quantity of probiotic bacteria flora, reduce human body intestinal canal pH value, improve human body intestinal canal microecological environment；High adsorption capacity, after modified, cellulosic specific surface area increases, and network is enriched, and absorption affinity strengthens, and the organic molecule ability of chelating, absorption cholesterol and bile acids is higher, suppress the human body absorption to them；Ion-exchange capacity strengthens, and to metallic element, particularly heavy metal element adsorption effect is higher, effectively prevent body weight for humans metal poisoning；Regulate and maintain the resident time of intestinal microbial population, strengthen digestion and the absorbability of intestinal, improve immunity of organisms；Effectively facilitate gastrointestinal peristalsis, slow down and eliminate the untoward reaction such as flatulence, abdominal distention；Powerful embedding effect can prevent environment (oxygen, temperature, illumination, the water activity etc.) factor impact on product quality, stabilizes the biological activity of bee pollen containing probiotic bacteria, extends the shelf-life of bee pollen containing probiotic bacteria.
7. the present invention is by composite to oligosaccharide and modified dietary fiber science, nutrient substance comprehensive, abundant is provided to probiotic bacteria, the maximization bred with functional metabolic product that maximizes not only achieving probiotic bacteria accumulates, and effectively improves mouthfeel and the stability of the finished product bee pollen containing probiotic bacteria.
8. the preparation method preparation method technique of the present invention is simple, easy to operate, low for equipment requirements, can industrialization and large-scale production, modified dietary fiber is finally mixed, has played its powerful embedding effect so that the character of product is more stable, the shelf-life is longer.
What it should be noted that the present invention bee pollen containing probiotic bacteria has the technical effect that the result that each component technical characteristic and method feature are mutually worked in coordination with, interacted, the not superposition of simple technical characteristic (component function), the combination of each component technical characteristic and the collaborative effect produced, considerably beyond the superposition of each monotechnics feature functionality and effect, have advanced preferably and practicality.
Detailed description of the invention
The present invention is described below by specific embodiment.Unless stated otherwise, technological means used in the present invention is method known in those skilled in the art.It addition, embodiment is interpreted as illustrative, and unrestricted the scope of the present invention, the spirit and scope of the invention are limited only by the claims that follow.To those skilled in the art, under the premise without departing substantially from spirit and scope of the present invention, the various changes or the changes that carry out the material component in these embodiments and consumption fall within protection scope of the present invention.
Embodiment one:
Prepared by raw material
1, the preparation of plant extract
Its preparation method is:
By Fructus Hordei Germinatus and Fructus Tritici aestivi 9:2 Homogeneous phase mixing in mass ratio, it is crushed to granularity 0.8mm, obtains pulverizing Fructus Hordei Germinatus；Then by Fructus Chaenomelis, Fructus Ananadis comosi, Fructus Fici in ultrasonic washing unit at power 200W, ultrasonic cleaning 8min when frequency 30KHz, drain, it is crushed to granularity 0.8mm under room temperature, and 8:2:1.5 Homogeneous phase mixing in mass ratio, the pulverizing Fructus Hordei Germinatus adding mixture quality 4 times obtains raw mixture, add the water of raw mixture quality 2 times, it is 3.5 with Fructus Citri Limoniae acid for adjusting pH value, at power 225W, microwave extraction is carried out when frequency 2000Hz, wherein, each microwave exposure total time 70s, carry out compartment irradiation: irradiation 10s, interval 10s, control temperature 30 DEG C, such irradiation 10 times, simultaneously at power 250W, ultrasonic assistant extraction is carried out when frequency 35KHz；Insulation 2h, then, carries out microwave extraction when power 300W, frequency 2000Hz, wherein, each microwave exposure total time 100s, compartment irradiation is carried out: irradiation 15s, interval 10s, controls temperature 50 C, such irradiation 10 times, simultaneously at power 400W, carry out ultrasonic assistant extraction when frequency 45KHz, be finally naturally cooling to room temperature, in electric field intensity 30kV/cm, burst length 500 μ s, carries out high-pressure pulse electric and extracts 18min when pulse frequency 250Hz；Extracting solution filters to obtain the first filtrate, adds the water rinsing of filtering residue 4 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:4 Homogeneous phase mixing in mass ratio, being evaporated to solid content is 30%, obtains plant extract；
In above-mentioned ultrasonic washing unit, cleanout fluid is the sodium bicarbonate solution of 0.4%.
2, the preparation of pupa worm intestinal extract
Its preparation method comprises the steps:
The drone pupa intestinal 4:2 in mass ratio of the queen bee nit intestinal of 3 ages in days and 12 ages in days is mixed, add the sericin peptide taken solution that mass percent is 10% of mixture quality 1.5 times, stir, put-20 DEG C of freezing 8min, broken, grind to form homogenate, add the biological demulsifying agent breakdown of emulsion 30min of its quality 0.04%, at 22000g, under 4 DEG C of conditions, centrifugal 25min, collects the middle level liquid after being centrifuged for the first time, then at 22000g, centrifugal 25min under 4 DEG C of conditions, collects the middle level liquid after second time is centrifuged, obtains pupa worm intestinal extract；
The pH value of above-mentioned sericin peptide taken solution is 6.5；
The quality group of above-mentioned biological demulsifying agent becomes: glycolipid class: lipopeptid class: cell wall is in conjunction with class=6:5:3；
Wherein the quality group of glycolipid class biological demulsifying agent becomes: rhamnolipid: alkyl polyglucoside=4:1.5.
3, the preparation of modified dietary fiber
The preparation method of modified dietary fiber, comprises the following steps:
By inulin, Semen Tritici aestivi fiber, Herba avenae fatuae fiber 6:3:2 Homogeneous phase mixing in mass ratio, add the water of its quality 5 times, room temperature 200W, 38KHz condition supersound extraction 13min, then at electric field intensity 30kV/cm, burst length 400 μ s, carries out high voltage pulse electric field processing 13min when pulse frequency 300Hz；It is 5.5 with breast acid for adjusting pH value, adds the enzyme of mixture quality 0.2%, in 50 DEG C of enzymolysis 34min；Enzymolysis solution filters, and filtrate reduced in volume, lyophilization to moisture are 6.5% namely obtain modified dietary fiber；
Above-mentioned enzyme is cellulase, xylanase, laccase, pectase 2:2:1.5:1.5 Homogeneous phase mixing in mass ratio.
4, the preparation of Lactobacillus plantarum powder
Lactobacillus plantarum powder is prepared with Lactobacillus plantarum CGMCCNO.11763 according to a conventional method for starting strain, and its viable bacteria content is: 9 × 1012cfu/g；The cryoprotective agent that wherein freeze drying process adopts is with the animal or plant containing antifreeze protein for raw material; prepared through high-pressure pulse electric extraction, ultrasonic assistant microwave extraction and compound enzyme enzymolysis, Lactobacillus plantarum powder can be effectively improved at freezing dry process Viable detection；
Protectant preparation method, comprises the steps:
Winter rye, Caulis et Folium Ammopiptanthi Mongolici, sharkskin collagen protein are respectively washed, drain, 9:4:3 Homogeneous phase mixing in mass ratio, add the lactic acid moistening 5h that pH value is 4.2 of mixed material quality 0.5 times, pulverize immediately after-20 DEG C of freezing 1.5h, freezing thickness of feed layer 4cm, ground product particle diameter 2mm, it is subsequently added into the water of ground product quality 15 times, it is 4.5 with breast acid for adjusting pH value, at electric field intensity 30kV/cm under room temperature, burst length 400 μ s, carries out high voltage pulse electric field processing 25min when pulse frequency 250Hz；Then carry out microwave irradiation and extraction 18min when power 225W in room temperature, simultaneously at power 250W, when frequency 35KHz, carry out ultrasonic assistant extraction；Add the compound enzyme of extracting solution quality 1.5%, in 50 DEG C of enzymolysis 40min；Enzymolysis solution filters, filtrate concentrates, low-temperature grinding to particle diameter is that namely 0.2mm obtains protective agent；
Above-mentioned compound enzyme is cellulase, protease, amylase, pectase, tannase 3:2:2:1.5:1.5 Homogeneous phase mixing in mass ratio.
Used by following example two to six, plant extract, pupa worm intestinal extract, modified dietary fiber, Lactobacillus plantarum powder are prepared by embodiment one.
Embodiment two:
A kind of Haematocoocus Pluvialls bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 165 parts, plant extract 20 parts, pupa worm intestinal extract 25 parts, modified dietary fiber 25 parts, oligosaccharide 11 parts, 8 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 4 parts, mango powder 4 parts, Haematocoocus Pluvialls 0.9 part；
Wherein bee pollen is Semen Sesami bee pollen；
The preparation method of a kind of Haematocoocus Pluvialls bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
2) bee pollen is put in the ultrasonic washing unit equipped with 0.4% sodium bicarbonate solution, in 200W under room temperature, 40KHz cleans 4min, rinsing, drain, the sericin peptide taken solution that mass percent is 16% soaks 6min, take out, put-20 DEG C of freezing 8min, broken, immediately at power 4Kw, frequency 2450MHz, 130 DEG C of microwave heating 6s of temperature, add the water of former bee pollen quality 4 times, it is 5 with breast acid for adjusting pH value, in electric field intensity 30kV/cm, burst length 500 μ s, high voltage pulse electric field processing 13min is carried out under pulse frequency 300Hz room temperature condition, then room temperature 400W, 40KHz condition supersound process 13min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 45 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 13min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution add oligosaccharide, 25% modified dietary fiber, fully dissolve 8min, it is naturally cooling to 43 DEG C, add 55% ferment at constant temperature 4h of Lactobacillus plantarum opaque amount, then with the ramp of 0.9 DEG C/min to 53 DEG C, add 45% ferment at constant temperature 2.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 43 DEG C with the speed of 0.7 DEG C/min, continue fermentation 0.8h, fermentation liquor 200 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 30min with hydroxyl isomaltulose, mango powder and Haematocoocus Pluvialls, speed of agitator 30r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 15min, speed of agitator 50r/min, namely sterile filling, sealing, packaging obtain the Haematocoocus Pluvialls bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to 99%, in Haematocoocus Pluvialls bee pollen, Lactobacillus plantarum viable count is up to 7.5 × 1011cfu/g。
Embodiment three:
A kind of fish oil bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 160 parts, plant extract 15 parts, pupa worm intestinal extract 23 parts, modified dietary fiber 23 parts, oligosaccharide 9 parts, 7 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 3 parts, mango powder 3 parts, 0.5 part of fish oil；
Wherein bee pollen is bee pollen of maize；
The preparation method of a kind of fish oil bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
2) bee pollen is put in the ultrasonic washing unit equipped with 0.3% sodium bicarbonate solution, in 200W under room temperature, 40KHz cleans 3min, rinsing, drain, the sericin peptide taken solution that mass percent is 13% soaks 5min, take out, put-18 DEG C of freezing 5min, broken, immediately at power 3Kw, frequency 2450MHz, 120 DEG C of microwave heating 5s of temperature, add the water of former bee pollen quality 3 times, it is 4 with breast acid for adjusting pH value, in electric field intensity 20kV/cm, burst length 400 μ s, high voltage pulse electric field processing 10min is carried out under pulse frequency 200Hz room temperature condition, then room temperature 400W, 40KHz condition supersound process 10min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 40 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 10min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution add oligosaccharide, 20% modified dietary fiber, fully dissolve 5min, it is naturally cooling to 40 DEG C, add 50% ferment at constant temperature 3h of Lactobacillus plantarum opaque amount, then with the ramp of 0.8 DEG C/min to 50 DEG C, add 40% ferment at constant temperature 1.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 40 DEG C with the speed of 0.6 DEG C/min, continue fermentation 0.5h, fermentation liquor 100 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 25min with hydroxyl isomaltulose, mango powder and fish oil, speed of agitator 20r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 12min, speed of agitator 40r/min, namely sterile filling, sealing, packaging obtain the fish oil bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to 98.5%, in fish oil bee pollen, Lactobacillus plantarum viable count is up to 8.5 × 1011cfu/g。
Embodiment four:
A kind of Lepidinm meyenii Walp bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 170 parts, plant extract 25 parts, pupa worm intestinal extract 27 parts, modified dietary fiber 27 parts, oligosaccharide 13 parts, 9 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 5 parts, mango powder 5 parts, pueraria root powder 0.8 part；
Wherein bee pollen is Brassica campestris L pollen；
The preparation method of a kind of Lepidinm meyenii Walp bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
2) bee pollen is put in the ultrasonic washing unit equipped with 0.5% sodium bicarbonate solution, in 200W under room temperature, 40KHz cleans 5min, rinsing, drain, the sericin peptide taken solution that mass percent is 18% soaks 7min, take out, put-22 DEG C of freezing 10min, broken, immediately at power 5Kw, frequency 2450MHz, 140 DEG C of microwave heating 7s of temperature, add the water of former bee pollen quality 5 times, it is 6 with breast acid for adjusting pH value, in electric field intensity 40kV/cm, burst length 600 μ s, high voltage pulse electric field processing 15min is carried out under pulse frequency 400Hz room temperature condition, then room temperature 400W, 40KHz condition supersound process 15min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 50 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 15min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution add oligosaccharide, 30% modified dietary fiber, fully dissolve 10min, it is naturally cooling to 45 DEG C, add 60% ferment at constant temperature 5h of Lactobacillus plantarum opaque amount, then with the ramp of 1.0 DEG C/min to 55 DEG C, add 50% ferment at constant temperature 3.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 45 DEG C with the speed of 0.8 DEG C/min, continuing fermentation 1h, fermentation liquor 300 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 35min with hydroxyl isomaltulose, mango powder and pueraria root powder, speed of agitator 40r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 18min, speed of agitator 60r/min, namely sterile filling, sealing, packaging obtain the Lepidinm meyenii Walp bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to 99.5%, in Lepidinm meyenii Walp bee pollen, Lactobacillus plantarum viable count is up to 6 × 1011cfu/g。
Embodiment five:
A kind of plant sterol bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 150 parts, plant extract 10 parts, pupa worm intestinal extract 20 parts, modified dietary fiber 20 parts, oligosaccharide 7 parts, 6 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 2 parts, mango powder 2 parts, plant sterol 0.5 part；
Wherein bee pollen is bee pollen of maize；
The preparation method of a kind of plant sterol bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
2) bee pollen is put in the ultrasonic washing unit equipped with 0.3% sodium bicarbonate solution, in 200W under room temperature, 40KHz cleans 5min, rinsing, drain, the sericin peptide taken solution that mass percent is 13% soaks 7min, take out, put-18 DEG C of freezing 10min, broken, immediately at power 3Kw, frequency 2450MHz, 140 DEG C of microwave heating 5s of temperature, add the water of former bee pollen quality 5 times, it is 4 with breast acid for adjusting pH value, in electric field intensity 40kV/cm, burst length 400 μ s, high voltage pulse electric field processing 10min is carried out under pulse frequency 400Hz room temperature condition, then room temperature 400W, 40KHz condition supersound process 15min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 40 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 15min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution add oligosaccharide, 20% modified dietary fiber, fully dissolve 10min, it is naturally cooling to 40 DEG C, add 60% ferment at constant temperature 3h of Lactobacillus plantarum opaque amount, then with the ramp of 1.0 DEG C/min to 50 DEG C, add 50% ferment at constant temperature 1.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 40 DEG C with the speed of 0.8 DEG C/min, continuing fermentation 1h, fermentation liquor 100 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 35min with hydroxyl isomaltulose, mango powder and plant sterol, speed of agitator 20r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 18min, speed of agitator 40r/min, namely sterile filling, sealing, packaging obtain the plant sterol bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to 99%, in plant sterol bee pollen, Lactobacillus plantarum viable count is up to 9 × 1011cfu/g。
Embodiment six:
A kind of Cordyceps militaris (L.) Link. bee pollen containing probiotic bacteria, mainly has the raw material of following parts by weight to prepare:
Bee pollen 180 parts, plant extract 30 parts, pupa worm intestinal extract 30 parts, modified dietary fiber 30 parts, oligosaccharide 15 parts, 10 parts of Lactobacillus plantarum powder, hydroxyl isomaltulose 6 parts, mango powder 6 parts, Cordyceps militaris (L.) Link. 0.3 part；
Wherein bee pollen is Brassica campestris L pollen；
The preparation method of a kind of Cordyceps militaris (L.) Link. bee pollen containing probiotic bacteria, comprises the steps:
1) each supplementary material is weighed according to formula；
2) bee pollen is put in the ultrasonic washing unit equipped with 0.5% sodium bicarbonate solution, in 200W under room temperature, 40KHz cleans 3min, rinsing, drain, the sericin peptide taken solution that mass percent is 18% soaks 5min, take out, put-22 DEG C of freezing 5min, broken, immediately at power 5Kw, frequency 2450MHz, 120 DEG C of microwave heating 7s of temperature, add the water of former bee pollen quality 3 times, it is 6 with breast acid for adjusting pH value, in electric field intensity 20kV/cm, burst length 600 μ s, high voltage pulse electric field processing 15min is carried out under pulse frequency 200Hz room temperature condition, then room temperature 400W, 40KHz condition supersound process 10min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 50 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 10min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution add oligosaccharide, 30% modified dietary fiber, fully dissolve 5min, it is naturally cooling to 45 DEG C, add 50% ferment at constant temperature 5h of Lactobacillus plantarum opaque amount, then with the ramp of 0.8 DEG C/min to 55 DEG C, add 40% ferment at constant temperature 3.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 45 DEG C with the speed of 0.6 DEG C/min, continue fermentation 0.5h, fermentation liquor 300 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 25min with hydroxyl isomaltulose, mango powder and Cordyceps militaris (L.) Link., speed of agitator 40r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 12min, speed of agitator 60r/min, namely sterile filling, sealing, packaging obtain the Cordyceps militaris (L.) Link. bee pollen containing probiotic bacteria；
Adopting broker wall bee pollen sporoderm-broken rate prepared by said method up to 99.5%, in Cordyceps militaris (L.) Link. bee pollen, Lactobacillus plantarum viable count is up to 8 × 1011cfu/g。
Experimental example seven eats the change of T-CHOL after Haematocoocus Pluvialls bee pollen
Selecting the adult 48 of T-CHOL 180mg/dl-250mg/dl, men and women half and half, is randomly divided into three groups；150 milliliters of mineral waters are drunk in first group of dinner every day；Second group of common commercially available Haematocoocus Pluvialls bee pollen 150g of dinner every day Instant Drinks, the Haematocoocus Pluvialls bee pollen 150g of the 3rd group of dinner every day Instant Drinks embodiment of the present invention 2, every day period eats same food, and food includes meat, egg, vegetable and fruit.Respectively at the blood of the previous day that experiment starts and the 20th, 40,60 days acquisition test persons, measuring the total cholesterol level in blood, result is table 1 such as:
Table 1: total cholesterol level testing result in blood
As seen from the above table after the Haematocoocus Pluvialls bee pollen of the Instant Drinks embodiment of the present invention 2, the content generation significant change of the T-CHOL in adult's blood.Compared with common commercially available Haematocoocus Pluvialls bee pollen, the content of the T-CHOL in adult's blood is significantly reduced by Haematocoocus Pluvialls bee pollen of the present invention, and the content of the T-CHOL in mineral water composition year human blood significantly increases, although commercially available Haematocoocus Pluvialls bee pollen decreases, but compared with product of the present invention, effect is not notable, it follows that the present invention adopts Haematocoocus Pluvialls bee pollen prepared by the specific bacterial strain with reduction cholesterol characteristic to have the health-care effect well reducing cholesterol.
It should be understood that the bee pollen containing probiotic bacteria prepared by embodiment of the present invention 3-6 has above-mentioned technique effect equally, between each embodiment, diversity is not notable.
Embodiment eight Haematocoocus Pluvialls bee pollen shelf-life implants living preparation of lactobacillus stable content test of the present invention
Take the Haematocoocus Pluvialls bee pollen of the embodiment of the present invention 2 preparation in room temperature 22-25 DEG C, store 3 months, 6 months, 12 months, 24 months, 36 months respectively and measure Lactobacillus plantarum viable bacteria content under ventilation condition, result is table 2 such as:
Table 2: shelf-life implants living preparation of lactobacillus content detection result
Result above shows: the storage stability of Haematocoocus Pluvialls bee pollen of the present invention is better, environment resistant (temperature, appropriateness, oxygen, illumination, moisture etc.) influence factor's ability is big, shelf-life Lactobacillus plantarum viable bacteria content loss rate maximum (36 months) is 15%, higher than existing like product viable bacteria content, loss rate is low, long shelf-life, reflects that other bioactive ingredients of Haematocoocus Pluvialls bee pollen has same storage stability simultaneously.
It should be understood that embodiment of the present invention 3-6 has above-mentioned experiment effect equally, between each embodiment and little with above-mentioned experiment effect diversity.
The sensory evaluation test of embodiment nine Haematocoocus Pluvialls bee pollen of the present invention
The Haematocoocus Pluvialls bee pollen that the embodiment of the present invention 2 is prepared by 24 personnel is invited to judge with the Haematocoocus Pluvialls bee pollen of commercially available two kinds of similar identical dates of manufacture, sense organ is given a mark, wherein specialty and each 12 of layman, professional is young, middle aged, each 4 of old age, men and women half and half, layman is juvenile, young, middle aged, each 3 of old age, and men and women half and half；Marking includes outward appearance (20 points), quality (25 points), local flavor (30 points), four aspects of mouthfeel (25 points), and marking personnel independently carry out, and are independent of each other, and judges result with guarantee accurate.Having added up judging result, equal score value takes approximation, retains integer, is specifically shown in table 3:
Table 3: sensory evaluation statistical result
Note: show significant difference (P < 0.05) with the different lowercase alphabet of mark in a line, the different capitalization of mark represents difference extremely notable (P < 0.01), indicates same letter and represents difference not notable (P > 0.05).
Result above shows, any one will be substantially better than commercially available Haematocoocus Pluvialls bee pollen to Haematocoocus Pluvialls bee pollen prepared by the present invention from outward appearance, quality, local flavor and mouthfeel, particularly outward appearance, local flavor and mouthfeel are fabulous, also are adapted for different age group, the consumer of different hierarchy of consumption eats simultaneously.
It should be understood that the bee pollen containing probiotic bacteria prepared by embodiment of the present invention 3-6 has above-mentioned experiment effect equally, between each embodiment and little with above-mentioned experiment effect diversity.
The embodiment ten Haematocoocus Pluvialls of the present invention probiotic test of bee pollen
The Haematocoocus Pluvialls bee pollen embodiment of the present invention 2 prepared, preparing into viable count with sterilized water is 2 × 1010The Lactobacillus plantarum solution of CFU/mL, saves backup in 4 DEG C；
1, the 10mL Lactobacillus plantarum solution kept of going bail for is injected in test tube 1, adopts ten times of stepwise dilutions to 10-8, take 1mL diluent on flat board, the MRS agar culture medium being cooled to 45 DEG C poured on flat board (sterilizing), shake up rapidly after sterilizing.Will be equipped with the test tube 2 of 10mL Lactobacillus plantarum solution again to be placed in 80-90 DEG C of water-bath and heat 15-25min, take the Lactobacillus plantarum solution after heating and carry out ten times of stepwise dilutions to 10-8, take 1mL diluent on flat board, the MRS agar culture medium being cooled to 45 DEG C poured into upper at flat board (sterilizing) and shake up rapidly after sterilizing.Finally the flat board before heating and after heating is all cultivated under 35 DEG C of conditions 24h, calculates the quantity before and after heating.
It is shown that Viable detection has reached more than 88%.
2, the resistance test of simulated gastric fluid and intestinal juice: the hydrochloric acid 16.4mL taking 100g/L adds distilled water diluting, make pH value respectively 1.5,2.5 and 3.5, take 100mL dilute hydrochloric acid solution, it is separately added into 1g pepsin, it is made fully to dissolve, obtaining simulated gastric fluid, microporous filter membrane degerming (0.22 μm) is standby.Taking potassium dihydrogen phosphate 6.8g, the 500mL that adds water makes dissolving, regulates pH value to 6.8 with 0.1moL/L sodium hydroxide solution；Separately taking trypsin 10g, the 100mL that adds water makes dissolving, after two liquid mixing, is diluted with water to 1000ml, obtains simulated intestinal fluid, and microporous filter membrane degerming (0.22 μm) is standby.Take the 1mL Lactobacillus plantarum solution kept to join in the simulated gastric fluid of 9mL (i.e. ten times of stepwise dilutions), and fully mix on the oscillator rapidly, be subsequently placed in 30-45 DEG C of quiescent culture 2-4h.Take out culture fluid respectively when 1h, 2h, 3h, 4h and count remaining viable count immediately, comparing with former viable count, it is shown that Viable detection is 98%.Then each 1mL of the culture fluid digesting different time it is taken in simulated gastric fluid, it is inoculated in the simulated intestinal fluid that 9mLpH value is 6.8 respectively, it is placed in 30-45 DEG C of quiescent culture 2-4h, and respectively 0,3,6,24h sampling, measure its viable count, comparing with former viable count, result shows that Viable detection is 99%.
3, simulating the resistance test of cholate: make the solution of 1g/L with pancreatin, and add the Fel Sus domestica salt of 0.8% in the solution, the NaOH adjustment pH with 10% is 8.0, then with 0.45 μm of micro-filtrate membrane filtration degerming.The Lactobacillus plantarum solution inoculum kept by 0.5mL is simulated in cholate to 4.5mL, obtains culture fluid, the viable count of counting remaining after cultivating 24h.By culture fluid in sterile saline ten times of stepwise dilutions to 10-8, and pour into MRS, it is subsequently placed in 35 DEG C of quiescent culture 24h.Result shows that Viable detection is 99%.
Above result of the test shows, Lactobacillus plantarum probiotic (thermostability, resistance to pH, bile tolerance) in Haematocoocus Pluvialls bee pollen of the present invention is stronger, being especially suitable for human intestines and stomach's environment, in simulated gastrointestinal environments, survival rate is big, can be effectively improved gastrointestinal function.
It should be understood that the bee pollen containing probiotic bacteria prepared by embodiment of the present invention 3-6 has above-mentioned experiment effect equally, between each embodiment and little with above-mentioned experiment effect diversity.
Embodiment 11 Haematocoocus Pluvialls of the present invention bee pollen mouse intestinal performance test
The Haematocoocus Pluvialls bee pollen embodiment of the present invention 2 prepared, preparing into viable count with sterilized water is 2 × 1010The Lactobacillus plantarum solution of CFU/mL, saves backup in 4 DEG C；
Choosing common Kunming white mice 60, male and female half and half, 18-20g, conventional word is supported.Therefrom random choose 40,9:00 gavage lincomycin hydrochloride 0.2mL every morning (20mg)/only, other as a control group, every day same time gavage equivalent sterile saline, continuous one week, prepare the mouse model of alteration of intestinal flora.Model group mouse diet declines, and dead and phenomenon of significantly suffering from diarrhoea does not occur, arranges soft excrement, and profile normal aqueous divides more, and bedding and padding are moist.By 40 alteration of intestinal flora mices, being randomly divided into 2 groups, one group 20 is only used as treatment group, the Lactobacillus plantarum solution 0.5ml (2 × 10 that every day, gavage was kept10Cfu/ml)/only, another 20 are only used as natural recovering group, every day same time gavage equivalent sterile saline, continuous two weeks.21 days whole experimental periods, observing growth and the defecation situation of white mice every day, weigh in the 8th, 21 days mices to Haematocoocus Pluvialls bee pollen treatment group and natural recovering group, calculate each group of weight average rate of increase, result is table 4 such as；Within every 5 days, surveying each group of stool in mice escherichia coli quantity, calculate average, result is table 5 such as.Take stool in mice and be about 0.1g, in aseptic operating platform, add 3 beades (adding 0.5mL diluent with 0.1g excrement sample), dilute and inoculate maconkey agar culture medium, calculate every gram of coliform count wet in just.
Table 4: mice Gain weight
Table 5: the situation of coliform count in stool in mice
Haematocoocus Pluvialls bee pollen treatment group Mouse Weight average rate of increase (67.96%) is significantly higher than natural recovering group (32.14%)；Feed solution rear intestinal escherichia coli quantity to be remarkably decreased, reduce by 97.07%, it is substantially less than natural recovering group (24.78%), show the rapid field planting in white mice intestinal of the Lactobacillus plantarum in Haematocoocus Pluvialls bee pollen of the present invention, form dominant microflora, and effectively suppress the growth and breeding of the pathogen such as escherichia coli, and resident time is long, continues, effectively improves intestinal performance.
It should be understood that the bee pollen containing probiotic bacteria prepared by embodiment of the present invention 3-6 has above-mentioned experiment effect equally, between each embodiment and little with above-mentioned experiment effect diversity.
The impact on immunity of organisms of embodiment 12 present invention bee pollen containing probiotic bacteria
1 experiment purpose
Test (mouse forced swimming) by exercise tolerance, verify the raising immunity of the present invention bee pollen containing probiotic bacteria, antifatigue effect.
2 experiment materials and reagent
2.1 for reagent thing:
The commercially available bee pollen (G1) containing probiotic bacteria；The commercially available bee pollen (G2) containing probiotic bacteria；Bee pollen (G3-G7) containing probiotic bacteria prepared by embodiment of the present invention 2-6.
2.2 reagent:
Liver/muscle glycogen testing cassete, builds up institute of biological products purchased from Nanjing；Concentrated sulphuric acid (AR), Nanjing Chemistry Reagent Co., Ltd.；Normal saline, Shangdong Changfu Jiejing Pharmaceutical Industry Co., Ltd..
3. laboratory animal
ICR mice, ♂, cleaning grade, body weight 18-22g, Ningxia Medical University's comparative medicine center provide, the free diet of mice during experiment.
4. key instrument
Aluminum swimming trunk (50cm × 50cm × 40cm), galvanized wire, low-temperature and high-speed centrifuge: 5804R type, Eppendrof company；Water-bath: DK-S26 type, the grand experimental facilities company limited of upper Nereid；Electronic scale: BS224S type, Sartorius company；Stopwatch, thermometer
5. experiment packet
5.1 dosage packets and given the test agent give the time and at random mice are divided into 8 groups, often group 10,1st group to the 7th group medicine giving G1～G7 respectively, 8th group is blank group, give isopyknic distilled water, the often every average daily gavage of group 1 time, gavage volume is 0.2ml/10g, gives given the test agent continuously 30 days.
5.2 sample preparations the 1st group to the 7th group: weigh 2.25g drug sample, be assigned to 150ml with distilled water；Blank group: distilled water 150ml.
6. experimental technique
After 6.1 swimming with a load attached to the body experiment last administration 30min, putting mice in swimming trunk, the depth of water is no less than 30cm, water temperature 25 ± 1 DEG C, the sheet lead of rat-tail root load 5% body weight, and record mice swimming started to the dead time, as mice swimming time.
After 6.2 mice serum carbamide measure last administration 30min, not swimming with a load attached to the body 90min in the water that temperature is 30 DEG C, eyeball blood sampling 0.5mL (being not added with anticoagulant) is plucked after rest 60min, put 4 DEG C of refrigerator 3h, after hemopexis, the centrifugal 15min of 2000r/min, takes serum and send clinical laboratory of Affiliated Hospital of Ningxia Medical University to detect.
After the mensuration last administration 30min of 6.3 hepatic glycogen, not swimming with a load attached to the body 90min in the water that temperature is 25 ± 1 DEG C, cervical dislocation puts to death mice, clean with normal saline, and with, after filter paper suck dry moisture, accurately weighing liver 100mg, hepatic glycogen detection kit detection Mouse Liver glycogen content.
Blood sampling after the mensuration last administration 30min of 6.4 blood lactase acid, does not then bear a heavy burden and stops after the water went swimming 10min that temperature is 30 DEG C.Lactic acid instrument assay method: respectively before swimming, each blood sampling 20 μ L add in 40 μ L rupture of membranes liquid after rest 20min after swimming, after swimming, fully vibration smudge cells lactic acid instrument measures immediately.(blood lactase acid area under curve=5 × (after front blood lactase acid value+3 × swimming of swimming the blood lactase acid value of 20min after blood lactase acid value+2 × swimming of 0min)
7. observation index walking weight load, blood lactase acid, carbamide, glycogen initial value
8. statistical method experimental data is usedRepresent, adopt t inspection to compare between organizing
9. experimental result
The impact on Mouse Weight of 9.1 present invention bee pollen containing probiotic bacteria
Each group mice is after giving G1～G9 medicine, before, in, post-weight is shown in shown in following table respectively, and each original body mass organizing mice and weightening finish body weight compare equal no difference of science of statistics (P > 0.05) with matched group, it was shown that G1～G9 medicine is all without obvious toxicity.Experimental result refers to table 6.
The original body mass of table 6 swimming with a load attached to the body experiment mice, mid-term body weight and terminate body weight
The impact on the mice burden swimming time of 9.2 present invention bee pollen containing probiotic bacteria
After per os gives mice G1～G7 medicine, G1～G2 medicine compares with blank group, the mice burden swimming time can be obviously prolonged, there is significant difference (P < 0.05), present invention bee pollen G3～G7 medicine containing probiotic bacteria compares with blank group, can significantly extend the mice burden swimming time, there is pole significant difference (P < 0.01), and be substantially better than G1～G2 medicine.The results detailed in Table 7.
The impact on the mice burden swimming time of the table 7 bee pollen containing probiotic bacteria
" * " p < 0.05vs blank；
" * * " p < 0.01vs blank；
9.3 present invention bee pollen containing probiotic bacteria is on the impact of blood lactase acid before and after mouse movement
After per os gives the bee pollen containing probiotic bacteria of the mice present invention, blood lactase acid area under curve after mouse movement is compared by present invention bee pollen G3～G7 medicine containing probiotic bacteria with matched group significant difference (P < 0.05), decrease though G1～G2 medicine group Mouse Blood lactic acid area under curve compares with matched group, but and no difference of science of statistics (P > 0.05).Result is in Table 8.
Table 8 present invention bee pollen containing probiotic bacteria is on the impact of blood lactase acid level before and after mouse movement
" * " p < 0.05vs blank；
The impact on Mouse Liver glycogen of 9.4 present invention bee pollen containing probiotic bacteria
After per os gives mice G1～G7 medicine, G1～G2 medicine compares with blank group, Mouse Liver glycogen content all has obvious rising, there is significant difference (P < 0.05), present invention bee pollen G3～G7 medicine containing probiotic bacteria compares with blank group, Mouse Liver glycogen content all has obvious rising, has pole significant difference (P < 0.01), and is substantially better than G1～G2 medicine.The results detailed in Table 9.
The impact on Mouse Liver glycogen content of table 9 present invention bee pollen containing probiotic bacteria
" * " p < 0.05vs blank；
" * * " p < 0.01vs blank；
The impact on mice serum carbamide of 9.5 present invention bee pollen containing probiotic bacteria
After per os gives mice G1～G7 medicine, G1～G2 medicine group compares with blank group, after mouse movement, serum urea content all has obvious reduction, there is significant difference (P < 0.05), present invention bee pollen G3～G7 medicine containing probiotic bacteria compares with blank group, after mouse movement, serum urea content all has obvious reduction, has pole significant difference (P < 0.01), and is substantially better than G1～G2 medicine.The results detailed in Table 10.
The impact on mice serum urea content of table 10 present invention bee pollen containing probiotic bacteria
" * " p < 0.05vs blank；
" * * " p < 0.01vs blank；
10. experiment conclusion
This experiment is tested mainly through mice burden swimming, and the deposit simultaneously detecting Mouse Liver glycogen observes the effect of the present invention bee pollen raising immunity containing probiotic bacteria, resisting fatigue.Preliminary Results shows below:
1, the G3～G7 of the present invention bee pollen containing probiotic bacteria all can extend the mice burden swimming time (P < 0.01), and effect is substantially better than the bee pollen containing probiotic bacteria of other G1～G2.
2, biochemistry detection aspect shows, the each dosage group of the G3～G7 of the present invention bee pollen containing probiotic bacteria all can reduce move after lactic acid content produced by glucose anerobic glycolysis in mice serum, compare with matched group and have significant difference (P < 0.05), and although the bee pollen containing probiotic bacteria of other G1～G2 also can reduce after motion lactic acid content produced by glucose anerobic glycolysis in mice serum, but compare with matched group, no difference of science of statistics (P > 0.05)；
3, each dosage group of the G3～G7 of the present invention bee pollen containing probiotic bacteria all can significantly improve the deposit (P < 0.01) of glycogen in mouse liver, and effect is substantially better than the bee pollen containing probiotic bacteria of other G1～G2；
4, metabolic arthritis model finds, the G3～G7 of the present invention bee pollen containing probiotic bacteria can significantly reduce the content (P < 0.01) of urea in serum after mice is swum, and effect is substantially better than other G1～G2 bee pollen containing probiotic bacteria；
11. conclusion
Above-mentioned experiment proves that the present invention bee pollen containing probiotic bacteria can significantly improve immunity of organisms, improve muscle power and the endurance of mice, the content of urea in serum and lactic acid after reduction mouse movement, and the deposit of glycogen in mouse liver can be significantly improved, contribute to alleviating the fatigue that sports load causes；The time that mice burden swimming to power exhausts can be extended.
Claims (10)
1. the bee pollen containing probiotic bacteria, prepared by the raw material mainly having following parts by weight: bee pollen 150-180 part, plant extract 10-30 part, pupa worm intestinal extract 20-30 part, modified dietary fiber 20-30 part, oligosaccharide 7-15 part, Lactobacillus plantarum powder 6-10 part, hydroxyl isomaltulose 2-6 part, mango powder 2-6 part；
The described bee pollen containing probiotic bacteria also includes one or more adjuvants of following parts by weight；Algae 0.6-1.2 part, corn oligopeptide powder 0.5-1 part, pueraria root powder 0.5-1 part, fish oil 0.2-0.8 part, plant sterol 0.2-0.8 part, phosphatidyl serine 0.1-0.7 part, oligochitosan 0.1-0.7 part, marine fishbone collagen oligopeptide powder 0.1-0.7 part, Chinese herbal medicine 0.1-0.6 part, Cordyceps militaris (L.) Link. 0.1-0.5 part；Soybean protein isolate 0.1-0.5 part；
Described Lactobacillus plantarum powder is to prepare according to a conventional method with Lactobacillus plantarum CGMCCNO.11763 for starting strain.
null2. the bee pollen containing probiotic bacteria as claimed in claim 1，It is characterized in that，The preparation method of described pupa worm intestinal extract，Comprise the steps: the drone pupa intestinal 3-5:1-3 in mass ratio mixing of the queen bee nit intestinal by 2-3 age in days and 11-12 age in days，Add the sericin peptide taken solution that mass percent is 8-12% of mixture quality 1-2 times，Stir，Put-18-22 DEG C of freezing 5-10min，Broken，Grind to form homogenate，Add the biological demulsifying agent breakdown of emulsion 20-40min of its quality 0.03-0.05%,At 18000-25000g，Centrifugal 20-30min under 4 DEG C of conditions，Collect the middle level liquid after being centrifuged for the first time，Then at 18000-25000g，Centrifugal 20-30min under 4 DEG C of conditions，Collect the middle level liquid after second time is centrifuged，Obtain pupa worm intestinal extract.
3. the bee pollen containing probiotic bacteria as claimed in claim 2, it is characterised in that the pH value of described sericin peptide taken solution is 6-7.
4. the bee pollen containing probiotic bacteria as claimed in claim 2, it is characterised in that the quality group of described biological demulsifying agent becomes: glycolipid class: lipopeptid class: cell wall is in conjunction with class=5-7:4-6:2-4.
5. the bee pollen containing probiotic bacteria as claimed in claim 4, it is characterised in that the quality group of described glycolipid class biological demulsifying agent becomes: rhamnolipid: alkyl polyglucoside=3-5:1-2.
6. the bee pollen containing probiotic bacteria as claimed in claim 1, it is characterised in that the preparation method of described plant extract is: by Fructus Hordei Germinatus and Fructus Tritici aestivi 8-10:1-3 Homogeneous phase mixing in mass ratio, is crushed to granularity 0.5-1mm, obtains pulverizing Fructus Hordei Germinatus；nullThen by Fructus Chaenomelis、Fructus Ananadis comosi、Fructus Fici in ultrasonic washing unit at power 200W、Ultrasonic cleaning 5-10min when frequency 30KHz，Drain，It is crushed to granularity 0.5-1mm under room temperature，And 7-9:1-3:1-2 Homogeneous phase mixing in mass ratio，The pulverizing Fructus Hordei Germinatus adding mixture quality 3-5 times obtains raw mixture，Add the water of raw mixture quality 1-3 times，It is 3-4 with Fructus Citri Limoniae acid for adjusting pH value，At power 150-300W、Microwave extraction is carried out when frequency 2000Hz，Wherein，Each microwave exposure total time 60-80s,Carry out compartment irradiation: irradiation 10s，Interval 10s，Control temperature 20-35 DEG C，Such irradiation 10 times，Simultaneously at power 200-300W,Ultrasonic assistant extraction is carried out when frequency 30-40KHz；Insulation 1-3h, then, microwave extraction is carried out when power 200-400W, frequency 2000Hz, wherein, each microwave exposure total time 90-105s, carry out compartment irradiation: irradiation 15s, interval 10s, controls temperature 40-60 DEG C, such irradiation 10 times, simultaneously at power 300-500W, carry out ultrasonic assistant extraction when frequency 40-50KHz, be finally naturally cooling to room temperature, in electric field intensity 25-35kV/cm, burst length 400-600 μ s, carries out high-pressure pulse electric and extracts 15-20min when pulse frequency 200-300Hz；Extracting solution filters to obtain the first filtrate, adds the water rinsing of filtering residue 4 times, filters to obtain the second filtrate, and by the first filtrate and the second filtrate 1:3-5 Homogeneous phase mixing in mass ratio, being evaporated to solid content is more than 20%, obtains plant extract.
7. the bee pollen containing probiotic bacteria as claimed in claim 1, it is characterized in that, the preparation method of described modified dietary fiber, comprise the following steps: by inulin, Semen Tritici aestivi fiber, Herba avenae fatuae fiber 5-7:2-4:1-3 Homogeneous phase mixing in mass ratio, add its water of quality 3-7 times, room temperature 100-300W, 35-40KHz condition supersound extraction 10-15min, then at electric field intensity 20-40kV/cm, burst length 300-500 μ s, carries out high voltage pulse electric field processing 10-15min when pulse frequency 200-400Hz；It is 4.5-6.5 with breast acid for adjusting pH value, adds the enzyme of mixture quality 0.1-0.3%, in 45-55 DEG C of enzymolysis 20-48min；Enzymolysis solution filters, and filtrate reduced in volume, lyophilization to moisture are that namely 5-8% obtains modified dietary fiber；
Described enzyme is cellulase, xylanase, laccase, pectase 1-3:1-3:1-2:1-2 Homogeneous phase mixing in mass ratio.
8. the bee pollen containing probiotic bacteria as claimed in claim 1, it is characterized in that, described Chinese herbal medicine is to prepare through micronizing with one or more in Flos Chrysanthemi, Flos Sophorae, Radix Ginseng, Bulbus Lilii, Radix Puerariae, Fructus Lycii, Herba Taraxaci, Rhizoma Dioscoreae, Poria, Fructus Hippophae, Flos Rosae Rugosae, Flos Lonicerae, Herba Menthae, Fructus Momordicae, Colla Corii Asini, Fructus Jujubae, Folium Mori, Fructus Momordicae charantiae, Agaricus blazei Murrill.
9. the preparation method of the bee pollen containing probiotic bacteria as claimed in claim 1, it is characterised in that comprise the steps:
1) each supplementary material is weighed according to formula；
null2) bee pollen is put in the ultrasonic washing unit equipped with 0.3-0.5% sodium bicarbonate solution，In 200W under room temperature、40KHz cleans 3-5min，Rinsing，Drain，The sericin peptide taken solution that mass percent is 13-18% soaks 5-7min，Take out，Put-18-22 DEG C of freezing 5-10min，Broken，Immediately at power 3-5Kw,Frequency 2450MHz,Temperature 120-140 DEG C of microwave heating 5-7s,Add the water of former bee pollen quality 3-5 times，It is 4-6 with breast acid for adjusting pH value，In electric field intensity 20-40kV/cm，Burst length 400-600 μ s,High voltage pulse electric field processing 10-15min is carried out under pulse frequency 200-400Hz room temperature condition，Then room temperature 400W、40KHz condition supersound process 10-15min obtains bee pollen pretreatment fluid；
3) bee pollen pretreatment fluid is warming up to 40-50 DEG C, is added thereto to plant extract and pupa worm intestinal extract, stirs, insulation, enzymolysis 10-15min, obtain bee pollen enzymolysis solution；
4) in bee pollen enzymolysis solution, add the modified dietary fiber of oligosaccharide, 20-30%, fully dissolve 5-10min, it is naturally cooling to 40-45 DEG C, add the 50-60% ferment at constant temperature 3-5h of Lactobacillus plantarum opaque amount, then with the ramp of 0.8-1.0 DEG C/min to 50-55 DEG C, add the 40-50% ferment at constant temperature 1.5-3.5h of Lactobacillus plantarum opaque amount, finally it is cooled to 40-45 DEG C with the speed of 0.6-0.8 DEG C/min, continue fermentation 0.5-1h, fermentation liquor 100-300 eye mesh screen filters, and namely filtrate obtain broker wall bee pollen through ultrafiltration, concentrating under reduced pressure, lyophilization；
5) broker wall bee pollen is added V-type blending tank Homogeneous phase mixing 25-35min with hydroxyl isomaltulose, mango powder and adjuvant, speed of agitator 20-40r/min, it is subsequently adding residue modified dietary fiber Homogeneous phase mixing 12-18min, speed of agitator 40-60r/min, namely sterile filling, sealing, packaging obtain the bee pollen containing probiotic bacteria.
null10. the preparation method of the bee pollen containing probiotic bacteria as claimed in claim 9，It is characterized in that，The preparation method of cryoprotective agent in described Lactobacillus plantarum powder preparation process，Comprise the steps: winter rye、Caulis et Folium Ammopiptanthi Mongolici、Sharkskin collagen protein is respectively washed、Drain，8-10:3-5:2-4 Homogeneous phase mixing in mass ratio，Add the lactic acid moistening 3-8h that pH value is 3.8-4.5 of mixed material quality 0.1-1 times,Pulverize immediately after-18--22 DEG C of freezing 1-2h，Freezing thickness of feed layer 3-5cm，Ground product particle diameter 0.5-3mm,It is subsequently added into the water of ground product quality 10-20 times，It is 3.5-5.5 with breast acid for adjusting pH value，At electric field intensity 25-35kV/cm under room temperature，Burst length 300-500 μ s,High voltage pulse electric field processing 20-30min is carried out when pulse frequency 200-300Hz；Then carry out microwave irradiation and extraction 15-20min when power 150-300W in room temperature, simultaneously at power 200-300W, when frequency 30-40KHz, carry out ultrasonic assistant extraction；Add the compound enzyme of extracting solution quality 1-2%, in 45-55 DEG C of enzymolysis 30-50min；Enzymolysis solution filters, filtrate concentrates, low-temperature grinding to particle diameter is that namely 0.1-0.3mm obtains protective agent；
Described compound enzyme is cellulase, protease, amylase, pectase, tannase 2-4:1-3:1-3:1-2:1-2 Homogeneous phase mixing in mass ratio.
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| CN111296848A (en) * | 2020-03-26 | 2020-06-19 | 山东正信生物科技股份有限公司 | Edible bacillus coagulans powder and preparation process thereof |
| CN113677216A (en) * | 2018-07-16 | 2021-11-19 | 朱利亚尼股份公司 | Microbiological method for producing bee bread |
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