CN104164368A - Production process of honeysuckle bacterium submerged fermentation nutrient solution - Google Patents
Production process of honeysuckle bacterium submerged fermentation nutrient solution Download PDFInfo
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- CN104164368A CN104164368A CN201410378461.2A CN201410378461A CN104164368A CN 104164368 A CN104164368 A CN 104164368A CN 201410378461 A CN201410378461 A CN 201410378461A CN 104164368 A CN104164368 A CN 104164368A
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Abstract
The invention discloses a production process of a honeysuckle bacterium submerged fermentation nutrient solution, belonging to the technical field of microbial engineering. The invention discloses a large-scale artificial fermentation nutrient solution production technology of honeysuckle bacteria. The large-scale artificial fermentation nutrient solution production technology is characterized by comprising the following steps: by adopting a pure strain of the honeysuckle bacteria, culturing by adopting a PAD culture medium, transforming into culture performed by adopting a liquid culture medium, performing culturing and amplification by using a seed tank, and transferring to a production tank according to an inoculation amount of 10% for achieving a maximum quantity of production components of the fermentation nutrient solution after 36-72 hours. The fermentation nutrient solution disclosed by the invention is rich in fungal polysaccharide, amino acid and various micro elements, and can be used as a nutritional supplement. In addition, the fermentation nutrient solution has an anti-inflammation effect which is similar to that of honeysuckle, can be used for treating symptoms such as pharyngitis, upper respiratory infection and the like, can be used for clearing and nourishing the lung and adjusting gastrointestinal functions, and can be independently used or compounded with other traditional Chinese medicines so as to benefit mankind.
Description
Technical field
The invention belongs to microbial engineering field, the submerged fermentation nutritive medium production technique that relates to a kind of Japanese Honeysuckle bacterium (formal name used at school: the lobate layer of currant bacterium Phylloporia ribis (Schumach.:Fr.) Ryvarden), belongs to the product that traditional Chinese medicine and modern biotechnology organically combine.
Background technology
Japanese Honeysuckle bacterium (the lobate layer of currant bacterium Phylloporia ribis (Schumach.:Fr.) Ryvarden) is a kind of medicinal fungi on the famous famous-region drug Japanese Honeysuckle plant of Shandong, among the people in locality have a long medicinal history, within 2002, is written into " Shandong Province's Chinese medicinal materials standard ".This bacterium entity is used for the treatment of the oral inflammations such as pharyngitis, laryngitis, tonsillitis, and clinical efficacy is remarkable.Pharmacological experiment study proves, this bacterium have anti-inflammatory, antiviral, anticancer, promote the biological activitys such as human body immunity, be fungi-medicine resource a kind of preciousness, that can substitute Japanese Honeysuckle antiinflammation.For this resource of deep exploitation, we have carried out strain separating purifying to it, have obtained the pure strain of this bacterium, and by the biotechnology of liquid submerged fermentation, reach extensive artificial object of producing Japanese Honeysuckle bacterium fermented nutritive liquid.
At present, only 8,000,000 kilograms of China's Japanese Honeysuckle annual production, and domestic and international market demand is more than 2,000 ten thousand kilograms, annual insufficiency of supply-demand reaches 12,000,000 kilograms.The Japanese Honeysuckle bacterium fermented nutritive liquid that the present invention produces, can substitute to a certain extent Japanese Honeysuckle and use as quick heat-clearing and detoxicating drug, to alleviate the situation of Japanese Honeysuckle imbalance between supply and demand anxiety.In addition the Japanese Honeysuckle bacterium fermented nutritive liquid composition that the present invention produces is abundant, is rich in after testing fungus polysaccharide, amino acid and various trace elements, can be used as a kind of nutritious supplementary.
Summary of the invention
The former bacterial classification of the present invention Japanese Honeysuckle bacterium used, through qualification, belongs to mycota, Basidiomycota, Holobasidiomycetes, Aphyllophorales, Xiu Ge pore fungi section fungi.Sporophore out-of-shape, is flats, fist shape, semicircle or cloud sheet shape.Cap surface irregularity, hard and tough, imbricate is arranged, and red dark reddish brown is to shallow maroon, and tarnish, has concentric annular rib and trickle fine hair, 1.5-4 × 3-7cm, thin edge or slightly blunt, dark cinnamon.Without stem, sporophore back side tawny, naked eyes are covered with the tiny mouth of pipe as seen, and the mouth of pipe is shallow liver brown to dun, sometimes the newborn sporophore of visible cluster.The visible bacterial context of section is cellulosic, brown color, the long 1-2mm of tube.It is faint yellow that spore is, spherical or wealthy avette, diameter 3 μ m.
We select to use PDA substratum, this bacterium has been carried out to strain separating purifying, obtain the pure strain of this bacterium, and test by liquid culture, tentatively determine that mycelial fermentative medium formula is wort (10Bx) 10%, peptone 0.3%, glucose 1%, sucrose 1%, corn steep liquor 0.5%, PH5.5-6.0.Adopt above substratum, cultivate 96 hours shake-flask culture mycelial yields for 27 DEG C and reach higher, can reach 1.5%, and fermented nutritive liquid recovery rate >=85% now.Establish the pilot scale fermentation technological process of production; Test tube slant bacterial classification---500ml triangular flask liquid seeds (loading amount 200ml)---5L serum bottle seed (loading amount 3L)---1 ton of fermentor tank (dress 500L)---filter press---liquid collecting---filling sterilizing---packaging warehouse-in.Pilot scale fermentation substratum and large productive culture base are; Wort (10Bx) 15%, sucrose 1%, glucose 1%, peptone 0.3%, corn steep liquor 0.5%, PH5.5-6.0, culture condition is: loading amount 50%, inoculum size 6-10%; 27 ± 1 DEG C of temperature, mixing speed 160rpm, 6 cubes ms/h of air flows, within 36～72 hours, mycelia yield just reaches the highest, can reach 1.6%, and fermented nutritive liquid recovery rate >=95% now.
For fermented nutritive liquid is tested and assessed, first fermented hypha is carried out to quality evalution, we by Syrups by HPLC the fat-soluble component Quantitative Determination of Ergosterol of Japanese Honeysuckle bacterium fermented hypha done measure and with atom entity in Quantitative Determination of Ergosterol contrast, result proves with Agilent1100LC high performance liquid chromatograph, Zorbax eclipse XDB C8, (150mm × 4.6mm) chromatographic column, methyl alcohol is moving phase, detection wavelength is 282nm, flow velocity 1.0ml/min.Typical curve is linear good at 0.1～0.5mg/ml, and minimum limitation is 0.1mg/ml, and RSD=2.88%, therefore be rich in ergosterol in Japanese Honeysuckle bacterium fermented nutritive liquid.
This bacterial classification has been preserved (Haidian District, Beijing City Institute of Microorganism, Academia Sinica at China Committee for Culture Collection of Microorganisms's common micro-organisms center; Postcode 100080); Classification And Nomenclature: the brown pore fungi of currant lobate layer bacterium (new systematics name) ring rib is former name Xanthochrous nilgheriensis (Phylloporia ribis (Schumach.:Fr.) Ryvarden)
Deposit number is; CGMCC No.1195, preservation date on July 22nd, 2004.
Brief description of the drawings:
Accompanying drawing is that Japanese Honeysuckle bacterium fermented nutritive liquid is produced technical process.
Embodiment:
The liquid submerged fermentation nutritive medium technical process of Japanese Honeysuckle bacterium
1, the cultivation situation on PDA substratum: the pure strain being separated to is transferred to a little, transfer on PDA substratum, cultivate three days bacterium colony circle, neat in edge for 30 DEG C, tiling, middle portion protuberance, white, cotton-shaped, densification, back side yellow, the radial rill of tool, diameter 2.7-3.2cm.Micro-Microscopic observation hyphae colorless, elongated, tool tabula, crisscross, multi-branched, diameter 2-2.5 μ m; The cell walls of hyphal cell is thin and transparent, and visible clamp connexion of mycelial growth initial stage is grown when the later stage, branch was more, and clamp connexion is not obvious.
2, liquid culture: potato is removed to eye and crust, be cut into the square fritter of 1cm, take 600 grams, the 3000ml that adds water heats with aluminum pot on electric furnace, after its boiling, keeps 30 minutes, then uses four layers of filtered through gauze, and filtrate is for subsequent use.Take in addition 60 grams of glucose, 6 grams of potassium primary phosphates, 1.5 grams, magnesium sulfate, 1.2 grams, urea, add after suitable quantity of water dissolving, mix with above-mentioned potato decocting liquid, be finally settled to 3000ml.
Get 3000ml triangular flask, after clean oven dry lets cool, every bottle adds above-mentioned nutrient solution 150ml, notes not making liquid adhere on bottle wall, and the continuous plug of upper cover, finally with kraft paper, bungee sealing.Before inoculation bacterial classification, packaged triangular flask entirety is placed under ultraviolet lamp and is sterilized 40 minutes.
3, the inoculation of mycelia and cultivation: get the good Japanese Honeysuckle bacterium of solid culture growing state mycelia in little triangular flask and inoculate, note rejecting the solid medium that agar is contained in bottom, the diameter of hypha body is approximately 1～2mm, make the size of got hypha body be consistent as far as possible, get 5 hypha bodies for every bottle.As front Feng Pinhou again, be placed on constant temperature oscillator and cultivate, keep 27 DEG C of temperature, amplitude is 2cm, concussion speed is 100rpm.
4, Mycelium culture condition optimizing; Determining of the temperature of shake-flask culture and incubation time, adopts 500ml triangular flask, loading amount 200ml, and shaking flask rotating speed 150rpm, under 30 DEG C of temperature condition, measures the yield of mycelium dry weight after fermentation.Show that thus mycelial suitable culture temperature is 27 DEG C, under 27 DEG C of conditions, the time of shake-flask culture is 96 hours.
5, fermentative production: mycelial fermentative medium formula is wort (10Bx) 10%, peptone 0.3%, glucose 1%, sucrose 1%, corn steep liquor 0.5%, PH5.5-6.0.
Adopt above substratum, cultivate 96 hours shake-flask culture mycelial yields for 27 DEG C and reach higher, can reach 1.5%, and fermented nutritive liquid recovery rate >=85% now.Having established pilot scale fermentation produces and technical process: test tube slant bacterial classification---500ml triangular flask liquid seeds (loading amount 200ml)---5 tons of fermentor tanks of 5L serum bottle seed (each loading amount 3L)---500L fermentor tank (loading amount 300L)---.Substratum is: wort (10Bx) 15%, sucrose 1%, glucose 1%, peptone 0.3%, corn steep liquor 0.5%, PH5.0-6.0.Culture condition is: loading amount 60%, inoculum size 10%; 27 DEG C of temperature, mixing speed 160rpm, 6 cubes ms/h of air flows, within 36 hours, mycelia yield just reaches the highest, can reach 1.6%, and fermented nutritive liquid recovery rate >=95% now.
6, the fermented liquid of fermentor tank and mycelia are proceeded to plate-and-frame filter press press filtration, and to powder lyophilize, pack after press filtration, the Co irradiation miscellaneous bacteria of going out is for subsequent use.Meanwhile concentrate and collect liquid after press filtration, and undertaken by aseptic pipeline processing mode filling, and then sterilizing, packaging warehouse-in.
Reference
[1].Lee?IK，Lee?JH，Yun?BS.Polychlorinated?compounds?with?PPAR-gamma?agonistic?effect?from?the?medicinalfungus?Phellinus?ribis[J].Bioorg?Med?Chem?Lett.2008Aug15；18(16)：4566
[2].Moon?DC，Hwang?KH，Choi?KR，et?al.Constituents?of?ceramide?of?a?native?mushroom，Phellinus?ribis?inKorea[J].Analytical?Science?and?Technology，1994，7：547～554.
[3].Moon?DC.Some?preoxysterols?and?ceramides?from“Phellinus?ribis”，a?Korean?wild?mushroom[J].AnalysisScience?Technol.，1995；8(4)：901-906
[4].MizunoT2002；Kang?&?Chen?2005
[5]. Xu Lingchuan, Zhang Lianghong, Lian Shiwen. " morchella submerged fermentation deep layer technique " patent of invention, the patent No.: ZL 2,010 10579919.2
[6]. Liu Yuhong, Xu Lingchuan, Wang Jianping. the research of currant leaf pore fungi strongly-acid chemical composition " time precious traditional Chinese medical science traditional Chinese medicines " 2006,17 (9)
[7]. Liu Yuhong, Xu Lingchuan, Wang Jianping. brown pore fungi belongs to medicinal fungi chemical composition and " Chinese materia medica academic periodical " 2003,21 (12) wanted in pharmacological research bun
[8]. Liu Yuhong, Xu Lingchuan, Wang Jianping. the research " Chinese medicinal materials " 2005,28 (11) of currant leaf pore fungi chemical composition
Claims (3)
1. by a Technology for fermentative Production Japanese Honeysuckle bacterium fermented nutritive liquid, it is characterized in that: fermentative medium formula is wort (10Bx) 10%, peptone 0.3%, glucose 1%, sucrose 1%, corn steep liquor 0.5%, PH5.5-6.0; Cultivate 96 hours shake-flask culture nutritive medium recovery rates for 27 DEG C and reach the highest, can reach 95%; The technological process of production: test tube slant bacterial classification---------------seeding tank is put in storage serum bottle seed triangular flask liquid seeds to produce tank fermentation by filter press---filling sterilizing---packaging;
Described Japanese Honeysuckle bacterium is the lobate layer of currant bacterium Phylloporia ribis (Schumach.:Fr.) Ryvarden, and its preserving number is: CGMCC No 1195.
2. a Japanese Honeysuckle bacterium claimed in claim 1 is through being accredited as the lobate layer of currant bacterium Phylloporia ribis (Schumach.:Fr.) Ryvarden, it is characterized in that: this bacterium is the bacterial classification obtaining from the separation and purification of Japanese Honeysuckle plant, and its preserving number is; CGMCC No 1195, the mycelia color of growing on solid medium is pure white and dense, and it is faint yellow producing fermentation gained nutritive medium, after gained mycelia drying and crushing, is pale powder.
3. a fermented nutritive liquid claimed in claim 1, is rich in the organic chemical compositions such as fungus polysaccharide, sterol, triterpene, chlorogenic acid, comprises active cells and active factor, realizes anti-inflammatory, antiviral, hypoglycemic, reducing blood-fat therapeutic action by immunomodulatory; Suppress tumour by transferring or activate human body " endogenous cancer-resisting substance "; As amino acid nutrient supplement or as anti-inflammatory, antiviral, antineoplastic folk prescription or composite side and extract, in order to various formulations such as the health drink for the treatment of pharyngitis, upper respiratory tract infection, kinds of tumors and be made into, oral liquid, injections.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106036305A (en) * | 2015-09-15 | 2016-10-26 | 廉士文 | Method for preparing beverage from submerged fermentation broth of Phylloporis ribis |
| CN106929464A (en) * | 2017-03-01 | 2017-07-07 | 济南大学 | A kind of method that utilization plant source cigarette water promotes sterol effective constituents accumulation in the lobate layer bacterium of currant |
| CN108835619A (en) * | 2018-06-06 | 2018-11-20 | 于晓 | A kind of throat-clearing throat-moistening food and preparation method thereof |
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| CN102191183A (en) * | 2010-12-09 | 2011-09-21 | 徐凌川 | Morchella submerged fermentation production process |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102191183A (en) * | 2010-12-09 | 2011-09-21 | 徐凌川 | Morchella submerged fermentation production process |
Non-Patent Citations (2)
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| 刘玉红 等: "茶藨子叶孔菌化学成分的研究", 《中药材》 * |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106036305A (en) * | 2015-09-15 | 2016-10-26 | 廉士文 | Method for preparing beverage from submerged fermentation broth of Phylloporis ribis |
| CN106929464A (en) * | 2017-03-01 | 2017-07-07 | 济南大学 | A kind of method that utilization plant source cigarette water promotes sterol effective constituents accumulation in the lobate layer bacterium of currant |
| CN108835619A (en) * | 2018-06-06 | 2018-11-20 | 于晓 | A kind of throat-clearing throat-moistening food and preparation method thereof |
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