CN116287040A - Process for synthesizing epsilon-polylysine by mixing and fermenting streptomycete and mould - Google Patents

Process for synthesizing epsilon-polylysine by mixing and fermenting streptomycete and mould Download PDF

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CN116287040A
CN116287040A CN202211398323.1A CN202211398323A CN116287040A CN 116287040 A CN116287040 A CN 116287040A CN 202211398323 A CN202211398323 A CN 202211398323A CN 116287040 A CN116287040 A CN 116287040A
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polylysine
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曾昕
朱洺志
泰宝艳
唐慧
苏志伟
李珊珊
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Huaibei Normal University
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Abstract

The invention discloses a process for synthesizing epsilon-polylysine by co-culturing streptomycete and mould, which is characterized in that streptomycete albilineans IFO14147 is used as epsilon-polylysine producing bacteria, other mould is used as inducing bacteria, and the two are co-cultured to synthesize epsilon-polylysine under the same culture medium and the same fermentation condition; in the co-culture process, a cation exchange resin bag is adopted to immerse fermentation liquor to control the concentration of epsilon-polylysine in the system, and the proper concentration of epsilon-polylysine is selected to control the growth of mould by utilizing the inhibition effect of epsilon-polylysine on mould, so that the mould continuously synthesizes a biological inducer which can promote the synthesis of epsilon-polylysine by streptomyces albicans, and finally, the fermentation yield is improved.

Description

Process for synthesizing epsilon-polylysine by mixing and fermenting streptomycete and mould
Technical Field
The invention relates to a method for promoting the synthesis of epsilon-polylysine, in particular to a method for mixing and culturing streptomyces albidoides and mould, which adopts the method for adjusting the submergence rate of cation exchange resin in fermentation liquor to ensure that the cation exchange resin can adsorb epsilon-polylysine exceeding the concentration range in the fermentation liquor, thereby regulating and controlling the growth of the mould according to the epsilon-polylysine concentration, further leading the mould to continuously synthesize microorganism signal molecules in the full fermentation stage so as to stimulate the streptomyces albidoides to efficiently synthesize epsilon-polylysine, and belongs to the technical field of industrial biology.
Background
Epsilon-polylysine is a homotype monomer polymer containing 25-35 lysine residues, and has the characteristics of wide bacteriostasis spectrum, good thermal stability, high-efficiency bacteriostasis and sterilization inhibition capability, green, safety and no toxicity. It was recognized by the U.S. FDA as a GRAS (global safety) compound and was approved by the national institutional committee as a food preservative for use in the food processing industry in 2014. Currently, epsilon-polylysine is mainly used in China, japan, the United states, the European Union, and Korea as a green and safe biological food preservative. In addition, epsilon-polylysine can be used as a drug carrier, a super absorbent polymer, a lipase inhibitor and a biochip for medical, health care and sanitary products, and has great market potential. The main reason for restricting the application of epsilon-PL at present is that the production cost is too high (1500 kg sold in the market) -1 ) Increasing the level of biosynthesis of epsilon-PL is an important means to solve this problem.
In this regard, a great deal of research work has been done in academia and industry, and the research has been focused mainly on the aspects of fine fermentation strain breeding, fermentation medium optimization, fermentation process regulation and control based on pH and oxygen supply, and the like. In the aspect of strain breeding, the current work mainly focuses on screening epsilon-PL high-yield strains which are tolerant to certain stress by a traditional mutation coupling cell fusion method; in the aspect of fermentation medium optimization, the current work mainly focuses on optimizing carbon and nitrogen sources in the medium or adding important intermediate metabolites and precursors in fermentation; in the aspect of fermentation process regulation, the industry mainly carries out regulation and control around the aspects of strengthening mass and oxygen transfer, dynamically regulating fermentation pH and the like. The main aim of the work is to improve the tolerance of the strain in fermentation and ensure the material energy requirement required by the synthesis of the product. However, there has been no corresponding research and development of related techniques for the fundamental problem of "why Streptomyces albus synthesizes epsilon-polylysine". Earlier studies of the subject group have found that the fungal cell extract greatly promotes the synthesis of epsilon-polylysine. The present subject group developed fermentation techniques based on the addition of a fungal extract by extracting some of the inducers from the fungal cells and adding them to epsilon-polylysine fermentation. However, this technique has its inherent bottleneck: (1) The culture process of the mould needs to consume culture medium, electric power and manpower, and brings corresponding organic fermentation wastewater; (2) The extraction process of the mould inducer is complicated, the organic solvent is consumed greatly, the energy is consumed for recovering the solvent, and the extra reagent and energy cost are brought; (3) The biological elicitor in the fungus extract can be continuously degraded by epsilon-polylysine producing bacteria, and the effect of activating epsilon-polylysine synthesis is mainly represented by 48 hours after addition, and the promotion effect in the middle and later stages of fermentation is not obvious.
The method for solving the problems is to perform co-culture fermentation on epsilon-polylysine producing bacteria (streptomyces albidoides) and inducer producing bacteria (mould) so that the mould continuously synthesizes the inducer and continuously activates epsilon-polylysine synthesizing capability of the streptomyces albidoides. However, epsilon-polylysine has a broad antibacterial spectrum, and at higher concentrations, can completely inhibit the growth of most bacteria, yeasts and molds; in turn, mold growth is much faster than streptomyces and has a resistance to epsilon-polylysine, which can grow rapidly in environments with no or low concentrations of epsilon-polylysine and dominate fermentation, making epsilon-polylysine non-synthetic.
The inventors have realized through a number of experiments and analyses that "regulating the epsilon-polylysine concentration in a fermentation broth" is a key to solving this problem. The invention adopts a mode of dynamically adding the cation exchange resin package to regulate and control the concentration of epsilon-polylysine in the fermentation liquor, so that the concentration can be in a proper range which just limits the rapid growth of mould and can not completely kill the mould. The method can lead the mould to be in proper abundance in the epsilon-polylysine fermentation broth so as to continuously synthesize the inducer, thereby obtaining the beneficial effect of continuously activating the Streptomyces albus to synthesize epsilon-polylysine. Compared with the traditional epsilon-polylysine fermentation and product in-situ extraction fermentation process, the method can obviously improve the product yield; compared with the fermentation of the fungus extract, the method omits the step of extracting the elicitor, and meanwhile, active fungus can continuously synthesize biological signal molecules to promote the synthesis of epsilon-polylysine, so that the method has important industrial application value.
Disclosure of Invention
In order to save the extraction link of the elicitor and continuously provide the biological elicitor capable of promoting the streptomyces albidoides to synthesize epsilon-polylysine, the invention takes the concentration of epsilon-polylysine in fermentation liquor as a regulating handle, adopts a dynamic immersion mode of a cation exchange resin package to regulate and control the concentration of epsilon-polylysine, further regulates and controls the growth rate of the mildew, so that the mildew and epsilon-polylysine generating bacteria keep a proper flora abundance ratio, thereby continuously synthesizing the biological elicitor and promoting the streptomyces albidoides to synthesize epsilon-polylysine for a long time. The method of the invention not only can obviously improve the fermentation yield of epsilon-polylysine, but also can greatly reduce the cost required by culturing mould cells, extracting inducers, treating related wastewater and controlling the precise fed-batch of inducers, and has important application value in epsilon-polylysine industrial production.
The invention discloses a process for synthesizing epsilon-polylysine by co-culturing streptomyces albulus IFO14147 (CICC 11022) serving as epsilon-polylysine producing bacteria and other various moulds serving as inducing bacteria, wherein the epsilon-polylysine is synthesized by co-culturing the streptomyces albulus IFO14147 and the other various moulds under the same culture medium and the same fermentation condition; in the co-culture process, a cation exchange resin bag is adopted to immerse fermentation liquor to control the concentration of epsilon-polylysine in the system, and the proper concentration of epsilon-polylysine is selected to control the growth of mould by utilizing the inhibition effect of epsilon-polylysine on mould, so that the mould continuously synthesizes a biological inducer which can promote the synthesis of epsilon-polylysine by streptomyces albicans, and finally, the fermentation yield is improved.
The invention discloses a process for synthesizing epsilon-polylysine by co-culturing streptomycete and mould, which comprises the following steps:
step 1: strain activation
Coating Streptomyces albus spores on a solid Bei Dana culture medium, and culturing at a constant temperature of 30 ℃ in a constant temperature incubator for 8-10 days until spores grow mature;
coating different mould spores on a PDA culture medium, and culturing at a constant temperature of 30 ℃ in a constant temperature incubator for 8-10 days until the spores grow to maturity;
the different mold comprises one or more of Penicillium chrysogenum, rhizopus oryzae, rhizopus niveus, rhizopus chinensis, aspergillus oryzae, monascus purpureus and Aspergillus niger.
Step 2: fermentation seed culture
2-3 ring Streptomyces albus spores (about 1X 10) 7 Inoculating in 500mL triangular flask containing 60mLM G culture medium, and culturing at 200rpm in a constant temperature shake incubator at 30deg.C for 1-2 days;
scraping 2-3 rings (about 1X 10) 7 And) inoculating different mould spores into a 500mL triangular flask with 80mLM G culture medium, and culturing for 1-3d at 30 ℃ in a constant temperature shaking incubator at 200 rpm.
Step 3: cation exchange resin modification
Placing cation exchange resin in a triangular flask, sequentially using 1mol/L ammonia water, 1mol/L hydrochloric acid and 1mol/L ammonia water solution which are 5 times of the volume of the resin, oscillating for 4 hours at 200rpm at a shaking table 30 ℃, and washing the resin with deionized water for many times until the pH value of a washing water solution is about 8.5 after each alkali acid-base modification; the modified cation exchange resin is wrapped by mesh cage media such as gauze or nylon bags to obtain a resin package, so that the resin can be conveniently added and taken out. The netpen media need to be sterilized prior to wrapping.
The cation exchange resin comprises Amberlite RC-50, HD-2, D004 or D152, etc., preferably Amberlite IRC-50.
Step 4: fermentation operation
Preparing M3G culture by using a 5L fermentation tank, sterilizing at 121 ℃ for 20min (glucose is independently sterilized), setting the total liquid loading amount of the reactor to 2-3L, inoculating 6-10% of the inoculated streptomyces albidoides fermentation seed liquid obtained in the step 2, and fermenting under the conditions of initial pH of 6.0-7.5 (preferably 6.8), temperature of 25-33 ℃ (preferably 29 ℃) and dissolved oxygen of 20-40% (preferably 30%; respectively inoculating the different mould fermentation liquor obtained in the step 2 with 10.0% -25% of inoculum size (preferably 13.3-20%) for fermentation for 0-60 h (preferably 24 h), filtering thallus, and controlling pH to 3.8-4.2 (preferably pH 4.0) with ammonia water until fermentation is completed; when the glucose concentration is lower than 10g/L, the concentration of the glucose solution in the fermentation liquor is maintained at 5-15g/L by supplementing 600g/L of glucose solution through a peristaltic pump; when ammonia nitrogen in the solution is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1.0g/L; when the epsilon-polylysine concentration is higher than 5g/L, the immersed operation of the resin bag is started to adsorb the redundant epsilon-polylysine, and the epsilon-polylysine in the fermentation tank is maintained at 4-7g/L, so that the concentration of the mould cells is kept constant. Fed-batch fermentation will end when the epsilon-polylysine concentration no longer increases.
The resin pack immersing operation needs to maintain a sterile environment, such as an operation in which the string holding the resin pack is released from the feeding port at the time of addition, or the like.
Step 5: re-release of epsilon-polylysine from resins
After the resin package obtained after fermentation was washed twice with deionized water, all the resins were disassembled and combined, washed twice with deionized water, and desorbed by shaking in a shaker at 30℃and 200rpm for 4 hours with 100ml of 0.8mol/L NaOH solution. After the end, the pH was adjusted back to 7.0 with 0.8mol/L HCl solution for the determination of the product concentration.
Step 6: sample detection
And detecting the concentration of epsilon-polylysine in the fermentation liquid: the fermentation broth was diluted appropriately with 0.2mM sodium phosphate buffer (ph=7.0), 2mL was taken out and mixed with 2mL of 1mM methyl orange aqueous solution, reacted at 30 ℃ for 30min, centrifuged at 4000rpm for 15min, the supernatant was mixed with 9mL of methanol, extracted with shaking in an ultrasonic cleaner for 20min, centrifuged at 4500g for 10min, the supernatant was diluted 20-fold with 0.2mM sodium phosphate buffer (ph=7.0), absorbance was measured at 465nm of spectrophotometer, and the actual epsilon-polylysine concentration was calculated by substituting into epsilon-polylysine concentration standard curve.
Detecting the concentration of glucose in fermentation liquor: 300. Mu.L of the fermentation broth was mixed with 700. Mu.L of absolute ethanol, allowed to stand for 1 hour, centrifuged to collect the supernatant, and subjected to HPLC detection after passing through a 0.45 μm filter. An organic acid test column (AminexHPX-87H, 300X 7.8mM; hercules, USA) was used, the mobile phase was set to 5mM sulfuric acid, the column temperature was controlled at 60℃and the amount of sample introduced was 10. Mu.L.
The dry weight of the cells (Driedcell weight, DCW) was measured by differential filter paper weighing. 10mL of the fermentation broth was taken out of the 5L fermenter, centrifuged for 10min at 4,500 Xg, the precipitate was washed twice with distilled water, suction-filtered with filter paper (Φ7cm, medium speed, SCRC) dried and weighed at 105℃beforehand, and then dried at 105℃until the weight was constant, and then weighed, and the difference in weight between front and rear was calculated.
The epsilon-polylysine producing strain of the present invention is Streptomyces albus IFO14147 (CICC 11022).
The different mold includes Penicillium chrysogenum CICC41585 (Penicillium chrysogenum CICC 41585), rhizopus oryzae CICC40468 (Rhizopus soryzaeCCC 40468), rhizopus nikoense CICC41346 (Rhizopus nigriensis CICC 41346), rhizopus chinensis CICC41505 (Rhizopus schinensisCICC 41505), aspergillus oryzae CICC2339 (Aspergillus oryzae CIICC 2339), monascus purpureus CICC41601 (Monascus purpeusCICC 41601), aspergillus niger CICC40102 (Aspergillus niger CICC 40102), and is preferably Aspergillus niger CICC40102.
The formula of the culture medium adopted by the invention comprises the following steps:
(1) Bei Dana Medium (g/L): glucose 10, yeast powder 1, peptone 2, agar 20, pH7.5;
(2) M3G Medium (G/L): glucose 60, yeast powder 5, (NH) 4 ) 2 SO 4 10，MgSO 4 ·7H 2 O0.5，K 2 HPO 4 ·3H 2 O0.8，KH 2 PO 4 1.36,FeSO 4 ·7H 2 O0.03，ZnSO 4 ·7H 2 O0.04，pH＝6.8。
(3) PDA medium (g/L): potato 200, glucose 20, agar 20, ph natural.
All the above components related to glucose are prepared by separating sugar from other components, sterilizing at 121deg.C for 20min, and mixing with the same system culture medium.
Compared with the traditional epsilon-polylysine liquid fermentation, the method for synthesizing epsilon-polylysine by co-culturing streptomycete and mould can obviously improve the yield and the production strength of epsilon-polylysine; compared with epsilon-polylysine fermentation added with a fungal elicitor, the invention has larger yield improvement amplitude, saves various costs related to extracting signal molecules in fermentation liquor/thalli, simplifies process regulation in fermentation, and is easier to apply in industrial production.
The invention will be better understood with reference to the following examples. The specific material ratios, process conditions, and results described in the examples are illustrative of the invention and should not be construed as nor limiting the invention which is described in detail in the claims.
Detailed Description
The technical scheme of the invention is further analyzed and illustrated by the following specific examples.
Example 1: mould screening capable of co-culturing with streptomyces albidogenus to synthesize epsilon-polylysine
The Streptomyces albus seeds obtained in the step 2 are inoculated into a pre-sterilized triangular flask containing 30mLM G culture medium in an inoculation amount of 8%, and are placed in a constant temperature shaking incubator at 29 ℃ for culturing for 24 hours. At this time, a sodium citrate buffer solution with a final concentration of 10g/L and a pH of 4.0 is added, the pH of the fermentation broth is regulated to 4.0, and the mycelia of the mature mould seed solution (obtained in the step 2) are filtered in a sterile environment, and each obtained mould is weighed (1 g) and added into a Streptomyces albus fermentation broth, and the moulds comprise Penicillium chrysogenum, rhizopus oryzae, rhizopus nigricans, rhizopus oryzae, monascus purpureus and Aspergillus niger. After the triangular flask is sealed, the flask is continuously placed in a constant temperature shaking incubator at 29 ℃ for culturing for 24 hours, and then the concentration of polylysine is measured, wherein the measured yields are 0.50g/L (no added mould), 0.53g/L (added 1.0g of penicillium chrysogenum), 0.50g/L (added 1.0g of rhizopus oryzae), 0.52g/L (added 1.0g of rhizopus niger), 0.51g/L (added 1.0g of rhizopus chinensis), 0.52g/L (added 1.0g of aspergillus oryzae), 0.50g/L (added 1.0g of monascus purpureus) and 0.63g/L (added 1.0g of aspergillus niger); if the polylysine concentration was measured after 48 hours of cultivation in a shaking incubator at a constant temperature of 29℃the yields were measured to be 0.90g/L (without mold), 1.39g/L (with 1.0g of P.chrysogenum), 1.20g/L (with 1.0g of Rhizopus oryzae), 0.93g/L (with 1.0g of Rhizopus nigella), 1.14g/L (with 1.0g of Rhizopus nigella), 1.08g/L (with 1.0g of Aspergillus oryzae), 0.83g/L (with 1.0g of monascus purpureus), 1.92g/L (with 1.0g of Aspergillus nigella). The data show that the beneficial effects of the mould co-culture can be reflected 48 hours after the mould is added, and the mould capable of promoting the biosynthesis of polylysine comprises penicillium chrysogenum, rhizopus chinensis, rhizopus oryzae, aspergillus oryzae and aspergillus niger, wherein the most obvious promotion effect is aspergillus niger.
Example 2: optimization of Aspergillus niger addition time
And (2) inoculating the Streptomyces albus seeds obtained in the step (2) into a pre-sterilized triangular flask filled with 30mLM G culture medium in an amount of 8%, culturing for 0h, 12h, 24h, 36h and 48h in a constant-temperature shaking incubator at 29 ℃, taking out the triangular flask, and adding aspergillus niger bodies in an ultra-clean bench. After 12h, sodium citrate buffer with a final concentration of 10g/L, pH4.0, was added to adjust the pH of the broth to 4.0. The mature aspergillus niger seed liquid is obtained through the step 2, 1g of the thallus is weighed after sterile filtration and added into the Streptomyces albus fermentation liquid, the sealed triangular flask is placed in a constant temperature shaking incubator at 29 ℃ for culturing for 48 hours, and then the polylysine concentration is measured, and the measured yields are respectively 0g/L (0 h for adding aspergillus niger), 1.2g/L (12 h for adding aspergillus niger), 2.9g/L (24 h for adding aspergillus niger), 2.0g/L (36 h for adding aspergillus niger) and 0.5g/L (48 h for adding aspergillus niger).
Example 3: aspergillus niger addition optimization in co-culture system
And (2) inoculating the Streptomyces albus seeds obtained in the step (2) into a pre-sterilized triangular flask containing 30mLM G culture medium in an inoculation amount of 8%, placing the triangular flask in a constant-temperature shaking incubator at 29 ℃ for culturing for 24 hours, taking out the triangular flask, and adding aspergillus niger bodies in an ultra clean bench. After 12h, sodium citrate buffer with a final concentration of 10g/L, pH4.0, was added to adjust the pH of the broth to 4.0. The mature Aspergillus niger seed liquid is obtained in the step 2, and 1g, 2g, 3g, 4g, 5g, 6g, 7g and 8g of the thallus is weighed after sterile filtration and added into Streptomyces albus fermentation broth, the sealed triangular flask is placed in a constant temperature shaking incubator at 29 ℃ for culturing for 48 hours, and then the polylysine concentration is measured, and the yields are respectively 0.9g/L (0 g), 2.9g/L (1 g), 3.1g/L (2 g), 3.2g/L (3 g), 3.3g/L (4 g), 3.4g/L (5 g), 3.3g/L (6 g), 3.3g/L (7 g) and 2.9g/L (8 g). From the above data, the most effective addition of Aspergillus niger to promote polylysine synthesis was found to be 4-6g/30mL, i.e., 13.3-20.0%.
Example 4: optimization of optimal temperature in co-culture system
Inoculating Streptomyces albus seeds obtained in the step 2 into pre-sterilized triangular flasks filled with 30mLM G culture medium at an inoculum size of 8%, respectively placing the triangular flasks in a constant temperature shaking incubator at 27 ℃, 28 ℃, 29 ℃,30 ℃, 31 ℃, 32 ℃, 34 ℃, 35 ℃, 36 ℃ and 37 ℃ for culturing for 24 hours, taking out the triangular flasks, and adding Aspergillus niger bodies in an ultra-clean bench. After 12h, sodium citrate buffer with a final concentration of 10g/L, pH4.0, was added to adjust the pH of the broth to 4.0. Mature Aspergillus niger seed liquid was obtained by step 2, and after aseptic filtration, 5g of Aspergillus niger was weighed into Streptomyces albus fermentation broth, and after sealing the flask, it was placed in a constant temperature shaking incubator at the corresponding temperature for further cultivation for 48 hours, after which the polylysine concentration was measured at 2.2g/L (27 ℃), 2.7g/L (28 ℃), 3.3g/L (29 ℃), 3.1g/L (30 ℃), 2.7g/L (31 ℃), 1.1g/L (32 ℃), 0.4g/L (33 ℃), 0.1g/L (34 ℃), 0g/L (35 ℃), 0g/L (36 ℃), respectively. The data show that the optimal co-culture temperature is 28-30 ℃, the activity of two bacteria is low when the temperature is too low, and the fermentation efficiency is low; the too high temperature promotes the activity of Aspergillus niger, and simultaneously damages the activity of Streptomyces albus, causing dysbacteriosis and making the product unable to be synthesized.
Example 5: optimum polylysine maintenance concentration optimization in co-culture system
Amberlite IRC-50 resin modification was performed according to step 3 to prepare an ammonium resin, resin encapsulation was performed in the manner of step 4, simple modification of the fermenter was completed under the direction of step 5, and fed-batch fermentation of polylysine was performed according to step 7, during which immersion of the resin encapsulation was performed according to step 6 to maintain the cohesive lysine concentration in the tank. The fermentation tank is modified by adopting the steps: the resin bag net cage is made of 304 stainless steel net, the lower end of the net cage is sealed by stainless steel net, and the upper end is opened. A5L liquid fermentation tank is selected, and a resin-coated net cage prepared in advance is fixed at 4 groups of baffles in the fermentation tank by welding or cotton ropes and stainless steel wires.
Amberlite eIRC-50 is placed in a triangular flask, ammonia water, hydrochloric acid and ammonia water solution with the volume of 1mol/L which is 5 times that of the resin are sequentially used, shaking is carried out for 4 hours at the temperature of 200r/min at the temperature of 30 ℃ of a shaking table, and deionized water is needed to wash the resin for many times after each alkali acid-alkali modification until the pH value of a washing water solution is about 8.5, so that the Amberlite eIRC-50 ammonium resin is prepared. Wrapping the modified cation exchange resin with nylon bags with proper size, and binding and sealing with cotton ropes. One end (end A) of a cotton rope is connected with more than 2-5 resin bags in series and is hung above a resin bag net cage of the fermentation tank, the other end (end B) of the cotton rope is led out of the fermentation tank from a feed supplementing opening, the cotton rope outside the fermentation tank is wrapped by a silica gel tube to prevent the infection of mixed bacteria, and the tail end (end B) of the cotton rope is fixed in a feed supplementing bottle. After the sterilization of the fermentation tank system is finished, 75% ethanol is poured into a sterile feeding bottle fixed with the cotton rope B end so as to submerge the cotton rope B end, and a sterile environment foundation is laid for the subsequent adjustment of the cotton rope length. When the concentration of epsilon-polylysine in the fermentation tank exceeds the specified concentration, the immersed operation of the resin bag is started. Opening the feeding bottle, and regulating the end B of the cotton rope by using sterile forceps to pay off, wherein the paying-off degree is based on the content of epsilon-polylysine which is just saturated and adsorbed and higher by the resin bag at the end A of the cotton rope. During paying off, the stirring function of the fermentation tank is closed to prevent the resin bag from deviating, so that the resin bag enters the resin net cage and is immersed in fermentation liquid. After the operation is finished, the end B of the cotton rope is fixed in the feeding bottle, and the feeding bottle is screwed.
Preparing M3G culture by adopting the slightly modified 5L fermentation tank, sterilizing at 121 ℃ for 20min (glucose is independently sterilized), setting the total liquid loading amount of the reactor to be 3L, inoculating 10% of the inoculation amount into the Streptomyces albus fermentation seed liquid obtained in the step 2, and fermenting under the conditions of initial pH6.8, temperature 29 ℃ and dissolved oxygen of 30%; inoculating the mould fermentation liquor filtering bacteria obtained in the step 2 with 18% of inoculation amount in fermentation for 24 hoursAfter that, ammonia water is used to control the pH value to 3.8-4.2 until the fermentation is finished; when the glucose concentration is lower than 10g/L, the concentration of the glucose solution in the fermentation liquor is maintained at 5-15g/L by supplementing 600g/L of glucose solution through a peristaltic pump; when ammonia nitrogen in the solution is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1g/L; setting an epsilon-polylysine maintenance gradient, respectively maintaining the epsilon-polylysine concentration in the range of 0-2g/L, 2-4g/L, 4-7g/L and 7-11g/L, and starting a resin bag immersing operation to adsorb redundant epsilon 1-polylysine when the epsilon 0-polylysine concentration is higher than the corresponding maintenance concentration, wherein fed-batch fermentation lasts for 168 hours. The final yield was the total yield of the whole tank (including the product in the fermentation broth and the product after resin desorption), and the final yield of each batch fermentation (including 0g/L (. Epsilon.2-polylysine maintenance concentration of 0-2 g/L), 19.3g/L (epsilon.3-polylysine maintenance concentration of 2-4 g/L), 67.3g/L (epsilon.4-polylysine maintenance concentration of 4-7 g/L), and 37.2g/L (epsilon-polylysine maintenance concentration of 7-11 g/L) respectively. The data show that the ultra-low concentration of epsilon-polylysine can cause the aspergillus niger to grow rapidly, and the later period becomes a main flora, so that epsilon-polylysine can not be synthesized, and the product accumulated in the earlier period can be degraded, so that fermentation can not be continued; the epsilon-polylysine maintains the concentration to be too high, so that the strong inhibition effect on the growth of the aspergillus niger is brought, the induction effect brought by mould is only reflected in the early fermentation stage, and the later aspergillus niger is gradually killed by the epsilon-polylysine, and the induction effect is not generated any more. Nevertheless, partial adsorption of polylysine has the effect of promoting product synthesis, which may be that adsorption can alleviate some of the product inhibition to promote epsilon-polylysine synthesis. This section shows that the concentration of epsilon-polylysine is controlled within the range of 4-7g/L by the adsorption of resin during fermentation, so that the abundance of Streptomyces-mold flora is kept balanced, and further biological signals are continuously synthesized to promote epsilon-polylysine synthesis.
Example 6: traditional polylysine fermentation process, product in-situ extraction fermentation process, mould fungus extraction and addition fermentation process and comparison of fermentation process effects
Adopts the traditional epsilon-polylysine fermentation process, namely preparing M3G culture, sterilizing for 20min based on 121 ℃ (glucose is singly killed)Bacteria), setting the total liquid loading amount of the reactor to be 3L, inoculating 10% of the inoculation amount into the Streptomyces albus fermentation seed liquid obtained in the step 2, fermenting under the conditions of initial pH6.8, temperature 29 ℃ and dissolved oxygen 30%, and controlling pH4.0 by ammonia water until the fermentation is finished (168 h); when the glucose concentration is lower than 10g/L, the concentration of the glucose solution in the fermentation liquor is maintained at 5-15g/L by supplementing 600g/L of glucose solution through a peristaltic pump; when ammonia nitrogen in the solution is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1g/L. The final epsilon-polylysine fermentation yield was 22.6g/L.
Adopting a polylysine in-situ extraction fermentation process, preparing M3G culture by using the slightly modified 5L fermentation tank in the step 5, sterilizing for 20min based on 121 ℃ (glucose is independently sterilized), setting the total liquid loading amount of a reactor to be 3L, inoculating the streptomyces albidovatus fermentation seed liquid obtained in the step 2 with 10% of inoculation amount, fermenting under the conditions of initial pH6.8, temperature 29 ℃ and dissolved oxygen 30%, and controlling pH4.0 by ammonia water until the fermentation is finished (168 h); when the glucose concentration is lower than 10g/L, the concentration of the glucose solution in the fermentation liquor is maintained at 5-15g/L by supplementing 600g/L of glucose solution through a peristaltic pump; when ammonia nitrogen in the solution is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1g/L; and (3) adding a large amount of Amberlite eIRC-50 ammonium resin treated in the steps (3) and (4) as an in-situ extraction carrier, adsorbing as much epsilon-polylysine synthesized by fermentation in a fermentation tank as possible, and finally obtaining the total yield of epsilon-polylysine of 32.4g/L.
The method comprises the steps of adopting a mould fungus extraction and fermentation process, namely firstly using an M3G culture medium to cultivate Aspergillus niger fungus, adopting 75% ethanol for crude extraction, centrifuging an extracting solution at 3000rpm, rotationally evaporating a supernatant, redissolving with water, and sterilizing at 121 ℃ for 20min. Preparing M3G culture, sterilizing at 121deg.C for 20min (sterilizing with glucose alone), setting the total reactor liquid content to 3L, inoculating 10% of the seed liquid obtained in step 2, fermenting at initial pH6.8 and 29 deg.C under dissolved oxygen condition of 30%, and controlling pH4.0 with ammonia water until fermentation is completed (168 h); and (3) adding the prefabricated mould fungus extract with the final concentration of 36g wet fungus/L into a fermentation tank at one time during fermentation for 24 hours. When (when)When the glucose concentration is lower than 10g/L, a peristaltic pump is used for supplementing 600g/L glucose solution to maintain the concentration in the fermentation liquor at 5-15g/L; when ammonia nitrogen in the solution is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1g/L, and the final total yield of epsilon-polylysine is 39.4g/L.
Using the optimal fermentation conditions in example 6, a total yield of 67.3g/L of epsilon-polylysine was finally obtained. The yield brought by the method is 3.0 times of the yield of the traditional epsilon-polylysine fermentation process, 2.1 times of the yield of the resin-added in-situ adsorption-non-co-culture fermentation process, and 1.7 times of the yield of the mould fungus extract added directly. Therefore, the fermentation process has obvious product lifting effect and great industrial application value.
While the invention has been described in terms of preferred embodiments, it is not intended to limit the invention, but various changes and modifications can be made by one skilled in the art without departing from the spirit and scope of the invention, and therefore, such changes and modifications as do not depart from the spirit of the invention are intended to be within the scope of the invention as claimed.

Claims (7)

1. A process for synthesizing epsilon-polylysine by co-culturing streptomycete and mould is characterized in that:
streptomyces albus IFO14147 is used as epsilon-polylysine producing bacteria, other various moulds are used as inducing bacteria, and the epsilon-polylysine is synthesized by co-culturing the two bacteria in the same culture medium and under the same fermentation condition; in the co-culture process, immersing fermentation liquor in a cation exchange resin bag to control the concentration of epsilon-polylysine in the system, and utilizing the inhibition effect of epsilon-polylysine on mould, selecting proper epsilon-polylysine concentration to control mould growth, so that the mould continuously synthesizes a biological inducer which can promote the synthesis of epsilon-polylysine by streptomyces albicans, and finally, the fermentation yield is improved;
the different mould comprises one or more of penicillium chrysogenum, rhizopus oryzae, rhizopus nigricans, rhizopus chinensis, aspergillus oryzae, monascus purpureus and aspergillus niger;
the cation exchange resin comprises Amberlite IRC-50, HD-2, D004 or D152.
2. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 1, which is characterized by comprising the following steps:
step 1: strain activation
Coating Streptomyces albus spores on a solid Bei Dana culture medium, and culturing at a constant temperature of 30 ℃ in a constant temperature incubator for 8-10 days until spores grow mature;
coating different mould spores on a PDA culture medium, and culturing at a constant temperature of 30 ℃ in a constant temperature incubator for 8-10 days until the spores grow to maturity;
step 2: fermentation seed culture
Scraping 2-3 loops of Streptomyces albus spores, inoculating the spores into a 500mL triangular flask filled with 60mL M3G culture medium, and culturing the spores in a constant-temperature shake incubator at 200rpm for 1-2 days at 30 ℃;
scraping 2-3 rings of different mould spores, inoculating the mould spores into a 500mL triangular flask filled with 80mLM G culture medium, and culturing for 1-3d at 200rpm in a constant-temperature shake incubator at 30 ℃;
step 3: cation exchange resin modification
Placing cation exchange resin in a triangular flask, sequentially using 1mol/L ammonia water, 1mol/L hydrochloric acid and 1mol/L ammonia water solution which are 5 times of the volume of the resin, oscillating for 4 hours at 200rpm at a shaking table 30 ℃, and washing the resin with deionized water for many times until the pH value of a washing water solution is 8.5 after each alkali acid-base modification; the modified cation exchange resin is wrapped by a net cage medium to obtain a resin package;
step 4: fermentation operation
Adopting a 5L fermentation tank to prepare M3G culture, sterilizing for 20min at 121 ℃, setting the total liquid loading amount of the reactor to be 2-3L, and inoculating the Streptomyces albus fermentation seed liquid obtained in the step 2 for fermentation; inoculating the different mould fermentation liquid obtained in the step 2 to filter thalli after fermentation for 0-60 h, and controlling pH to 3.8-4.2 by ammonia water until fermentation is finished; when the concentration of the epsilon-polylysine in the system is higher than 5g/L, starting the immersing operation of the resin bag to adsorb the redundant epsilon-polylysine, and maintaining the concentration of the epsilon-polylysine in the fermentation tank at 4-7g/L.
3. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 2 wherein:
in the step 4, the inoculation amount of the fermentation seed liquid of the streptomyces albilineans is 6-10%.
4. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 2 wherein:
in the step 4, fermentation conditions after inoculating Streptomyces albus fermentation seed liquid are as follows: the initial pH value is 6.0-7.5, the temperature is 25-33 ℃, and the dissolved oxygen is 20-40%.
5. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 2 wherein:
in the step 4, the inoculation amount of the filtering thalli of different mould fermentation liquor is 10% -25%.
6. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 5 wherein the process is characterized by:
and (3) inoculating the different mould fermentation liquid obtained in the step (2) to the fermentation tank for 48h to filter the thalli.
7. The process for synthesizing epsilon-polylysine by co-culturing streptomycete and mold according to claim 2 wherein:
in the fermentation process, when the glucose concentration of the system is lower than 10g/L, a peristaltic pump is used for supplementing 600g/L of glucose solution to maintain the concentration of the fermentation liquid at 5-15g/L; when the ammonia nitrogen concentration of the system is lower than 0.5g/L, 40 percent (NH) of the pre-sterilization is fed through an external source 4 ) 2 SO 4 So as to maintain the ammonia nitrogen concentration to be 0.5-1.0g/L.
CN202211398323.1A 2022-11-09 2022-11-09 Process for synthesizing epsilon-polylysine by mixing and fermenting streptomycete and mould Pending CN116287040A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116768971A (en) * 2023-07-31 2023-09-19 浙江昊清生物科技有限公司 Method for producing thiostrepton by microbial fermentation
CN116768971B (en) * 2023-07-31 2024-05-14 浙江昊清生物科技有限公司 Method for producing thiostrepton by microbial fermentation

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