A process for synthesizing ε-polylysine through mixed fermentation of Streptomyces and molds.

Abstract

This invention discloses a process for the co-culture synthesis of ε-polylysine by Streptomyces and molds. Streptomyces IFO14147 is used as the ε-polylysine producing bacterium, and various other molds are used as inducing bacteria. Both are co-cultured under the same culture medium and fermentation conditions to synthesize ε-polylysine. During the co-culture process, the fermentation broth is immersed in cation exchange resin to control the concentration of ε-polylysine in the system. Utilizing the inhibitory effect of ε-polylysine on molds, an appropriate ε-polylysine concentration is selected to control mold growth. This allows the molds to continuously synthesize biological inducers that promote the synthesis of ε-polylysine by Streptomyces IFO14147, ultimately resulting in increased fermentation yield.

Description

Technical Field

[0001] This invention relates to a method for promoting ε-polylysine synthesis. Specifically, it involves co-culturing *Streptomyces leucovorum* and other molds, and adjusting the immersion rate of cation exchange resin in the fermentation broth to allow the cation exchange resin to adsorb ε-polylysine exceeding its concentration range in the fermentation broth. This allows the ε-polylysine concentration to regulate mold growth, thereby enabling the mold to continuously synthesize microbial signaling molecules throughout the entire fermentation process to stimulate *Streptomyces leucovorum* to efficiently synthesize ε-polylysine. This method belongs to the field of industrial biotechnology. Background Technology

[0002] ε-Polylysine (PPL) is a homopolymer containing 25-35 lysine residues. It possesses a broad antibacterial spectrum, good thermal stability, high bactericidal and antimicrobial activity, and is green, safe, and non-toxic. It has been designated GRAS (Generally Recognized As Safe) by the US FDA and was approved by my country's National Health and Family Planning Commission in 2014 for use as a food preservative in the food processing industry. Currently, PPL is primarily used as a green and safe biological food preservative in China, Japan, the US, the EU, and South Korea. Furthermore, PPL can also be used as a drug carrier, superabsorbent polymer, lipase inhibitor, and biochip in pharmaceuticals, health products, and hygiene products, showing significant market potential. The main reason currently limiting the application of PPL is its high production cost (commercially available at ¥1500 kg). -1 Improving the biosynthesis level of ε-PL is an important means to solve this problem.

[0003] Extensive research has been conducted in academia and industry on this issue, primarily focusing on the selection of superior fermentation strains, optimization of fermentation media, and pH and oxygen supply-based fermentation process regulation. In strain selection, current work mainly focuses on screening for high-yielding ε-PL strains tolerant to certain stresses using traditional mutagenesis-coupled cell fusion methods. Regarding fermentation media optimization, current work focuses on optimizing carbon and nitrogen sources in the media or adding important intermediate metabolites and precursors during fermentation. In fermentation process regulation, industry efforts mainly revolve around enhancing mass and oxygen transfer and dynamically adjusting fermentation pH. The main goal of this work is to improve the strains' tolerance during fermentation and ensure the necessary material and energy requirements for product synthesis. However, no corresponding research or related technology development has been established to address the fundamental question of "why *Streptomyces cepacia* synthesizes ε-polylysine." Our previous research found that mold cell extracts can significantly promote ε-polylysine synthesis. Our group extracted inducers from some mold cells and added them to ε-polylysine fermentation, developing a fermentation technology based on the addition of mold cell extracts. However, this technology has its inherent bottlenecks: (1) The cultivation process of mold requires the consumption of culture medium, electricity and labor, and at the same time brings corresponding organic fermentation wastewater; (2) The extraction process of mold inducers is complicated, the consumption of organic solvents is large, and the recovery of solvents requires energy, which brings additional reagent and energy costs; (3) The biological inducers in the mold cell extract will be continuously degraded by ε-polylysine producing bacteria, and its role in activating ε-polylysine synthesis is mainly manifested in the 48 hours after addition, and the promotion effect in the middle and late stages of fermentation is not obvious.

[0004] The solution to the above problems lies in co-culturing and fermenting ε-polylysine-producing bacteria (Streptomyces albopictus) and inducer-producing bacteria (mold). This allows the mold to continuously synthesize inducers and persistently activate the ε-polylysine synthesis capacity of Streptomyces albopictus. However, ε-polylysine has a broad antibacterial spectrum; at high concentrations, it can completely inhibit the growth of most bacteria, yeasts, and molds. Conversely, molds grow much faster than Streptomyces and exhibit some resistance to ε-polylysine. In environments without or with low concentrations of ε-polylysine, molds can grow rapidly and dominate fermentation, preventing the synthesis of ε-polylysine.

[0005] Through extensive experimentation and analysis, the inventors recognized that "regulating the concentration of ε-polylysine in the fermentation broth" is key to solving this problem. This invention employs a dynamic addition of cation exchange resin packets to regulate the concentration of ε-polylysine in the fermentation broth, ensuring it remains within a suitable range that limits rapid mold growth without completely killing the mold. This method allows for optimal mold abundance in the ε-polylysine fermentation broth, enabling continuous synthesis of inducers and thus achieving the beneficial effect of continuously activating *Streptomyces leucovorum* to synthesize ε-polylysine. Compared to traditional ε-polylysine fermentation and in-situ product extraction fermentation processes, this method significantly increases product yield. Compared to fermentation with added mold cell extracts, this method eliminates the inducer extraction step, while the active mold continuously synthesizes biosignal molecules to promote ε-polylysine synthesis, possessing significant industrial application value. Summary of the Invention

[0006] To eliminate the need for inducer extraction and continuously provide bio-inducers that promote ε-polylysine synthesis by *Streptomyces leucovorum*, this invention uses the ε-polylysine concentration in the fermentation broth as a control mechanism. It employs a dynamic immersion method with cation exchange resin packs to regulate the ε-polylysine concentration, thereby controlling the mold growth rate and maintaining an optimal ratio of mold to ε-polylysine-producing bacteria. This ensures continuous synthesis of bio-inducers and long-term promotion of ε-polylysine synthesis by *Streptomyces leucovorum*. This method not only significantly increases ε-polylysine fermentation yield but also substantially reduces the costs associated with mold cell culture, inducer extraction, wastewater treatment, and precise feedstock control of the inducer. It has significant application value in the industrial production of ε-polylysine.

[0007] This invention relates to a process for the co-culture synthesis of ε-polylysine using Streptomyces albulus IFO14147 (CICC11022) as the ε-polylysine producing bacterium and various other molds as inducing bacteria. Both are co-cultured under the same culture medium and fermentation conditions to synthesize ε-polylysine. During co-culture, the fermentation broth is immersed in cation exchange resin to control the ε-polylysine concentration. The inhibitory effect of ε-polylysine on molds is utilized, and an appropriate ε-polylysine concentration is selected to control mold growth. This allows the molds to continuously synthesize biological inducers that promote ε-polylysine synthesis in Streptomyces albulus, ultimately leading to increased fermentation yield.

[0008] The process for synthesizing ε-polylysine through streptomyces-mold co-culture of the present invention includes the following steps:

[0009] Step 1: Activation of microbial strains

[0010] Spread the spores of Streptomyces albopictus on solid betanna medium and incubate them at 30°C for 8-10 days until the spores mature.

[0011] Different fungal spores were spread on PDA medium and cultured at 30°C for 8-10 days in a constant temperature incubator until the spores matured.

[0012] The different molds include one or more of Penicillium chrysogenum, Rhizopus oryzae, Rhizopus nigricans, Rhizopus huassifolius, Aspergillus oryzae, Monascus purpureus, and Aspergillus niger.

[0013] Step 2: Fermentation of Seed Culture

[0014] Scrape 2-3 rings of Streptomyces spores (approximately 1×10⁻⁶) 7 Each flask was inoculated into a 500mL Erlenmeyer flask containing 60mL of LM3G medium and incubated at 30℃ and 200rpm for 1-2 days in a constant temperature shaking incubator.

[0015] Scrape 2-3 rings (approximately 1×10) 7 Different fungal spores were inoculated into 500mL Erlenmeyer flasks containing 80mL LM3G medium and cultured at 30℃ and 200rpm in a constant temperature shaking incubator for 1-3 days.

[0016] Step 3: Cation exchange resin modification

[0017] The cation exchange resin was placed in an Erlenmeyer flask, and then sequentially treated with 5 times the resin volume of 1 mol / L ammonia, 1 mol / L hydrochloric acid, and 1 mol / L ammonia solution. The mixture was shaken on a shaker at 30°C and 200 rpm for 4 hours. After each alkali-acid-alkali modification, the resin was washed multiple times with deionized water until the pH of the washing solution was approximately 8.5. The modified cation exchange resin was then wrapped in a mesh medium such as gauze or nylon bags to obtain a resin package, facilitating the addition and removal of the resin. The mesh medium required sterilization before wrapping.

[0018] The cation exchange resin includes Amberlite IRC-50, HD-2, D004 or D152, etc., preferably Amberlite IRC-50.

[0019] Step 4: Fermentation process

[0020] A 5L fermenter was used. M3G culture medium was prepared and sterilized at 121℃ for 20 min (glucose was sterilized separately). The total liquid volume of the reactor was set to 2-3L. The *Streptomyces simonii* fermentation seed culture obtained in step 2 was inoculated at a rate of 6-10%. Fermentation was carried out under the following conditions: initial pH 6.0-7.5 (preferably 6.8), temperature 25-33℃ (preferably 29℃), and dissolved oxygen 20%-40% (preferably 30%). Different mold fermentation broths obtained in step 2 were inoculated at a rate of 10.0%-25% (preferably 13.3-20%) between 0-60 hours (preferably 24 hours) of fermentation, and the cells were filtered. The culture was then treated with ammonia water. Maintain the pH at 3.8-4.2 (preferably pH 4.0) until fermentation is complete. When the glucose concentration falls below 10 g / L, add 600 g / L of glucose solution via a peristaltic pump to maintain the concentration in the fermentation broth at 5-15 g / L. When the ammonia nitrogen concentration in the solution falls below 0.5 g / L, add pre-sterilized 40% (NH4)2SO4 externally to maintain the ammonia nitrogen concentration at 0.5-1.0 g / L. When the ε-polylysine concentration exceeds 5 g / L, initiate the resin pack immersion process to adsorb excess ε-polylysine, maintaining the ε-polylysine concentration in the fermenter at 4-7 g / L, at which point the mold cell concentration will remain constant. Fed-batch fermentation will end when the ε-polylysine concentration no longer increases.

[0021] The immersion of resin packs requires a sterile environment, such as adding resin by releasing the cotton rope securing the resin pack through the filling tube.

[0022] Step 5: Re-release of ε-polylysine from the resin

[0023] After fermentation, the resin packets were washed twice with deionized water. All resins were then combined and washed twice more with deionized water. 100 mL of 0.8 mol / L NaOH solution was added, and the mixture was shaken at 30°C and 200 rpm for 4 hours for desorption. After desorption, the pH was adjusted to 7.0 with 0.8 mol / L HCl solution for product concentration determination.

[0024] Step 6: Sample Testing

[0025] Detection of ε-polylysine concentration in fermentation broth: The fermentation broth was appropriately diluted with 0.2 mM sodium phosphate buffer (pH = 7.0), and 2 mL of the diluted solution was mixed with 2 mL of 1 mM methyl orange aqueous solution. The mixture was placed at 30℃ for 30 min, centrifuged at 4000 rpm for 15 min, and the supernatant was mixed with 9 mL of methanol. The mixture was then extracted by shaking in an ultrasonic cleaner for 20 min, centrifuged at 4500 g for 10 min, and the supernatant was diluted 20 times with 0.2 mM sodium phosphate buffer (pH = 7.0). The absorbance was measured at 465 nm using a spectrophotometer, and the actual ε-polylysine concentration was calculated by substituting the absorbance into the ε-polylysine concentration standard curve.

[0026] Glucose concentration detection in fermentation broth: 300 μL of fermentation broth was mixed with 700 μL of anhydrous ethanol, allowed to stand for 1 h, centrifuged, and the supernatant was filtered through a 0.45 μm filter membrane before HPLC detection. An organic acid test column (Aminex HPX-87H, 300 × 7.8 mm; Hercules, USA) was used, with the mobile phase set to 5 mM sulfuric acid, the column temperature controlled at 60 °C, and the injection volume 10 μL.

[0027] Dry cell weight (DCW) was determined using the filter paper differential weighing method. 10 mL of fermentation broth was taken from a 5 L fermenter, centrifuged at 4,500 × g for 10 min, and the precipitate was washed twice with distilled water. The precipitate was then filtered through pre-dried and weighed filter paper (Φ7 cm, medium speed, SCRC). The filter paper was then dried at 105 °C until constant weight and weighed. The weight difference before and after the initial measurement was calculated.

[0028] In this invention, the ε-polylysine-producing bacterium is Streptomyces albulus IFO14147 (CICC11022).

[0029] The different molds included Penicillium chrysogenum CICC41585, Rhizopus oryzae CICC40468, Rhizopus nigricans CICC41346, Rhizopus chinensis CICC41505, Aspergillus oryzae CICC2339, Monascus purpureus CICC41601, and Aspergillus niger CICC40102, all purchased from the China Industrial Microbial Culture Collection Center (CICC), with Aspergillus niger CICC40102 being the preferred variety.

[0030] The culture medium formulation used in this invention is as follows:

[0031] (1) Betana medium (g / L): glucose 10, yeast extract 1, peptone 2, agar 20, pH 7.5;

[0032] (2) M3G medium (g / L): glucose 60, yeast extract 5, (NH4)2SO4 10, MgSO4·7H2O 0.5, K2HPO4·3H2O 0.8, KH2PO4 1.36, FeSO4·7H2O 0.03, ZnSO4·7H2O 0.04, pH=6.8.

[0033] (3) PDA medium (g / L): potato 200, glucose 20, agar 20, pH natural.

[0034] All of the above-mentioned glucose-containing substances were prepared separately from other components, sterilized at 121°C for 20 minutes, and then combined into the same culture medium system.

[0035] Compared with traditional liquid fermentation of ε-polylysine, the method of synthesizing ε-polylysine by co-culturing Streptomyces and molds in this invention can significantly improve the yield and production intensity of ε-polylysine. Compared with ε-polylysine fermentation with the addition of fungal inducers, this invention has a greater yield increase and eliminates the costs involved in extracting signal molecules from fermentation broth / cells, simplifies process control in fermentation, and is easier to apply in industrial production.

[0036] The present invention can be better understood by referring to the specific embodiments below. The specific material ratios, process conditions, and results described in the embodiments are only for illustrating the present invention and should not and will not limit the present invention as described in detail in the claims. Detailed Implementation

[0037] The technical solution of the present invention will be further analyzed and explained through specific embodiments below.

[0038] Example 1: Screening of molds capable of synthesizing ε-polylysine by co-culturing with Streptomyces albopictus

[0039] The *Streptomyces simonii* seeds obtained in step 2 were inoculated at an 8% inoculation rate into pre-sterilized Erlenmeyer flasks containing 30 mL of LM3G medium and incubated in a 29°C constant temperature shaking incubator for 24 hours. At this time, sodium citrate buffer solution with a final concentration of 10 g / L (pH 4.0) was added to adjust the pH of the fermentation broth to 4.0. The mature mold seed culture (obtained in step 2) was then filtered under aseptic conditions. The obtained mold cells (1 g each) were weighed and added to the *Streptomyces simonii* fermentation broth. These molds include *Penicillium chrysogenum*, *Rhizopus oryzae*, *Rhizopus nigricans*, *Rhizopus huassifolius*, *Aspergillus oryzae*, *Monascus purpureus*, and *Aspergillus niger*. Afterwards, the Erlenmeyer flasks were sealed and incubated in a 29℃ constant temperature shaking incubator for 24 hours. The polylysine concentration was then measured, and the yields were: 0.50 g / L (without mold), 0.53 g / L (with 1.0 g Penicillium chrysogenum), 0.50 g / L (with 1.0 g Rhizopus oryzae), 0.52 g / L (with 1.0 g Rhizopus nigricans), 0.51 g / L (with 1.0 g Rhizopus huassifolius), 0.52 g / L (with 1.0 g Aspergillus oryzae), 0.50 g / L (with 1.0 g purple yeast), and 0.63 g / L (with 1.0 g...). (Aspergillus niger); after culturing in a constant temperature shaking incubator at 29℃ for 48 hours, the polylysine concentration was measured, and the yields were 0.90 g / L (without mold), 1.39 g / L (with 1.0 g Penicillium chrysogenum), 1.20 g / L (with 1.0 g Rhizopus oryzae), 0.93 g / L (with 1.0 g Rhizopus niger), 1.14 g / L (with 1.0 g Rhizopus schlegelii), 1.08 g / L (with 1.0 g Aspergillus oryzae), 0.83 g / L (with 1.0 g Monascus purpureus), and 1.92 g / L (with 1.0 g Aspergillus niger). The above data indicate that the beneficial effects of mold co-culture are evident 48 hours after mold addition. The molds that can promote polylysine biosynthesis include Penicillium chrysogenum, Rhizopus schlegelii, Rhizopus oryzae, Aspergillus oryzae, and Aspergillus niger, with Aspergillus niger showing the most significant promoting effect.

[0040] Example 2: Optimization of Aspergillus niger addition time

[0041] The *Streptomyces simonii* seeds obtained in step 2 were inoculated at an 8% inoculum into pre-sterilized Erlenmeyer flasks containing 30 mL of LM3G medium. The flasks were then incubated at 29°C with a shaking incubator for 0 h, 12 h, 24 h, 36 h, and 48 h. Afterward, *Aspergillus niger* cells were added to the flasks in a clean bench. After 12 h, sodium citrate buffer solution with a final concentration of 10 g / L (pH 4.0) was added to adjust the pH of the fermentation broth to 4.0. The mature Aspergillus niger seed culture was obtained in step 2. After aseptic filtration, 1g of the mycelium was weighed and added to the Streptomyces simulans fermentation broth. The Erlenmeyer flask was sealed and placed in a constant temperature shaking incubator at 29℃ for 48h. The polylysine concentration was then measured, and the yields were 0g / L (0h Aspergillus niger added), 1.2g / L (12h Aspergillus niger added), 2.9g / L (24h Aspergillus niger added), 2.0g / L (36h Aspergillus niger added), and 0.5g / L (48h Aspergillus niger added). The above data show that the optimal timing for adding Aspergillus niger mycelium is 24h during Streptomyces simulans fermentation.

[0042] Example 3: Optimization of Aspergillus niger addition in co-culture system

[0043] The *Streptomyces simonii* seeds obtained in step 2 were inoculated at an 8% inoculum into a pre-sterilized Erlenmeyer flask containing 30 mL of LM3G medium. The flask was then incubated in a 29°C shaking incubator for 24 hours. Afterward, *Aspergillus niger* cells were added to the flask in a clean bench. After 12 hours, sodium citrate buffer solution with a final concentration of 10 g / L (pH 4.0) was added to adjust the pH of the fermentation broth to 4.0. Mature Aspergillus niger seed culture was obtained in step 2. The mycelium was aseptically filtered and weighed at 1g, 2g, 3g, 4g, 5g, 6g, 7g, and 8g, and added to the Streptomyces simonii fermentation broth. The Erlenmeyer flasks were sealed and incubated at 29℃ for 48 hours. The polylysine concentration was then measured, and the yields were 0.9g / L (0g), 2.9g / L (1g), 3.1g / L (2g), 3.2g / L (3g), 3.3g / L (4g), 3.4g / L (5g), 3.3g / L (6g), 3.3g / L (7g), and 2.9g / L (8g), respectively. Based on these data, the most effective Aspergillus niger addition for promoting polylysine synthesis is 4-6g / 30mL, i.e., 13.3-20.0%.

[0044] Example 4: Optimization of the optimal temperature in the co-culture system

[0045] The *Streptomyces simonii* seeds obtained in step 2 were inoculated at an 8% inoculation rate into pre-sterilized Erlenmeyer flasks containing 30 mL of LM3G medium. The flasks were then incubated for 24 hours in constant temperature shaking incubators at 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 34°C, 35°C, 36°C, and 37°C, respectively. Afterward, *Aspergillus niger* cells were added to the flasks in a clean bench. After 12 hours, sodium citrate buffer solution with a final concentration of 10 g / L (pH 4.0) was added to adjust the pH of the fermentation broth to 4.0. Mature Aspergillus niger seed culture was obtained in step 2. After aseptic filtration, 5g of Aspergillus niger mycelium was weighed and added to the fermentation broth of Streptomyces simulans. The Erlenmeyer flask was sealed and placed in a constant temperature shaking incubator at the appropriate temperature for 48h. The polylysine concentration was then measured, and the yields were 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℃), and 0g / L (37℃). The data above indicates that 28-30℃ is the optimal co-culture temperature. If the temperature is too low, the activity of both bacteria will be at a low level, resulting in low fermentation efficiency. If the temperature is too high, the activity of Aspergillus niger will be increased, while the activity of Streptomyces albopictus will be damaged, causing dysbiosis and preventing the synthesis of the product.

[0046] Example 5: Optimization of the optimal polylysine maintenance concentration in the co-culture system

[0047] Following step 3, AmberliteIRC-50 resin was modified to prepare ammonium-type resin. Resin encapsulation was performed using the method described in step 4. A simple modification of the fermenter was completed under the guidance of step 5. Polylysine was then fed in batches and fermented according to step 7, with the resin packs submerged as described in step 6 to maintain the polylysine concentration within the tank. The fermenter modification involved fabricating resin pack cages using 304 stainless steel mesh, sealing the bottom of the cage with stainless steel mesh and leaving the top open. A 5L liquid fermenter was selected, and the pre-prepared resin pack cages were fixed to four sets of baffles inside the fermenter using welding or cotton rope and stainless steel wire.

[0048] AmberliteIRC-50 was placed in an Erlenmeyer flask, and then 1 mol / L ammonia, hydrochloric acid, and ammonia solution (5 times the resin volume) were added sequentially. The mixture was shaken on a shaker at 30°C and 200 rpm for 4 hours. After each alkali-acid-alkali modification, the resin was washed multiple times with deionized water until the pH of the washing solution was around 8.5, thus preparing AmberliteIRC-50 ammonium-type resin. The modified cation exchange resin was wrapped in a nylon bag of appropriate size and sealed with cotton rope. Two to five or more resin bags were connected in series at one end (end A) of a cotton rope and suspended above the resin bag cage in the fermenter. The other end (end B) of the cotton rope exited the fermenter through the feed inlet. The cotton rope outside the fermenter was wrapped with silicone tubing to prevent contamination. The end of the cotton rope (end B) was fixed in the feed bottle. After the fermenter system was sterilized, 75% ethanol was poured into the sterile feed bottle with end B of the cotton rope fixed in place to submerge end B, establishing a sterile environment for subsequent adjustments to the rope length. When the ε-polylysine concentration in the fermenter exceeds the specified concentration, initiate the resin pack immersion operation. Open the feed bottle and use sterile tweezers to adjust end B of the cotton rope for threading. The threading depth should be such that the resin pack at end A of the cotton rope can just saturate and adsorb the excess ε-polylysine content. During threading, turn off the fermenter's stirring function to prevent the resin pack from deviating, ensuring it enters the resin mesh cage and is immersed in the fermentation broth. After the operation is complete, secure end B of the cotton rope in the feed bottle and tighten the feed bottle.

[0049] Using a slightly modified 5L fermenter, M3G culture medium was prepared and sterilized at 121℃ for 20 min (glucose was sterilized separately). The total liquid volume of the reactor was set to 3L. A 10% inoculum of the *Streptomyces albopictus* fermentation seed culture obtained in step 2 was added, and fermentation was carried out under the conditions of initial pH 6.8, temperature 29℃, and dissolved oxygen 30%. After 24 hours of fermentation, an 18% inoculum of the *Streptomyces albopictus* fermentation broth obtained in step 2 was added, and the cells were filtered. The pH was then controlled at 3.8-4.2 with ammonia until fermentation was complete. When the glucose concentration fell below 10 g / L, 600 mg / L glucose was added via a peristaltic pump. The concentration of glucose in the fermentation broth was maintained at 5-15 g / L. When the ammonia nitrogen in the solution was below 0.5 g / L, pre-sterilized 40% (NH4)2SO4 was added externally to maintain the ammonia nitrogen concentration at 0.5-1 g / L. An ε-polylysine maintenance gradient was set to maintain the ε-polylysine concentration in the ranges of 0-2 g / L, 2-4 g / L, 4-7 g / L, and 7-11 g / L, respectively. When the ε-polylysine concentration was higher than the corresponding maintenance concentration, the resin pack immersion operation was started to adsorb the excess ε-polylysine. The fed-batch fermentation lasted for 168 h. The final yield is the total yield of the entire fermentation tank (including products in the fermentation broth and products after resin desorption). The final yields (inclusive) for each batch of fermentation are 0 g / L (ε-polylysine maintenance concentration 0-2 g / L), 19.3 g / L (ε-polylysine maintenance concentration 2-4 g / L), 67.3 g / L (ε-polylysine maintenance concentration 4-7 g / L), and 37.2 g / L (ε-polylysine maintenance concentration 7-11 g / L). Data shows that too low an ε-polylysine maintenance concentration will lead to rapid growth of Aspergillus niger, which will become the dominant microorganism in the later stages. It will not only be unable to synthesize ε-polylysine, but will also degrade the products accumulated in the early stages, making fermentation unsustainable. Too high an ε-polylysine maintenance concentration will have a strong inhibitory effect on the growth of Aspergillus niger. The inducing effect of the mold is only reflected in the early stage of fermentation. In the later stages, Aspergillus niger is gradually killed by ε-polylysine and no longer exerts an inducing effect. Nevertheless, partial adsorption of polylysine still promotes product synthesis, possibly because adsorption can alleviate some of the inhibitory effects on the product, thereby promoting ε-polylysine synthesis. This section shows that using resin adsorption to control the ε-polylysine concentration within the range of 4-7 g / L during fermentation can maintain a balance in the abundance of the Streptomyces-mold community, thereby continuously synthesizing biosignals to promote ε-polylysine synthesis.

[0050] Example 6: Comparison of the effects of traditional polylysine fermentation process, in-situ product extraction fermentation process, mold cell extraction and addition fermentation process, and the fermentation process of the present invention.

[0051] The traditional ε-polylysine fermentation process was employed, involving the preparation of M3G culture medium sterilized at 121℃ for 20 min (glucose sterilized separately). The total reactor volume was set to 3L. A 10% inoculum of *Streptomyces albopictus* fermentation seed culture obtained in step 2 was inoculated. Fermentation was carried out under initial conditions of pH 6.8, temperature 29℃, and dissolved oxygen 30%, with pH controlled at 4.0 using ammonia water until fermentation was completed (168h). When the glucose concentration fell below 10g / L, 600g / L glucose solution was added via a peristaltic pump to maintain the concentration in the fermentation broth at 5-15g / L. When the ammonia nitrogen concentration in the solution fell below 0.5g / L, pre-sterilized 40% (NH4)2SO4 was added externally to maintain the ammonia nitrogen concentration at 0.5-1g / L. The final ε-polylysine fermentation yield was 22.6g / L.

[0052] The polylysine in-situ extraction and fermentation process was adopted. A slightly modified 5L fermenter (as described in step 5) was used. M3G culture medium was prepared and sterilized at 121℃ for 20 min (glucose was sterilized separately). The total liquid volume of the reactor was set to 3L. 10% of the *Streptomyces cylindrica* fermentation seed culture obtained in step 2 was inoculated. Fermentation was carried out under the conditions of initial pH 6.8, temperature 29℃, and dissolved oxygen 30%, with pH controlled at 4.0 using ammonia water until the end of fermentation (168 h). When the glucose concentration was below 10 g / L, peristalsis was used to control the fermentation. The pump replenished 600 g / L of glucose solution to maintain the concentration in the fermentation broth at 5-15 g / L; when the ammonia nitrogen in the solution was lower than 0.5 g / L, pre-sterilized 40% (NH4)2SO4 was added externally to maintain the ammonia nitrogen concentration at 0.5-1 g / L; a large amount of AmberliteIRC-50 ammonium resin treated in steps 3 and 4 was added as an in-situ extraction carrier to adsorb as much ε-polylysine synthesized in the fermenter as possible, and the final total yield of ε-polylysine was 32.4 g / L.

[0053] A fungal mycelium extraction and fermentation process was employed. First, *Aspergillus niger* cells were cultured in M3G medium. After crude extraction with 75% ethanol, the extract was centrifuged at 3000 rpm, the supernatant was rotary evaporated, and the extract was reconstituted with water and sterilized at 121°C for 20 min. M3G medium was prepared and sterilized at 121°C for 20 min (glucose was sterilized separately). The total volume of the reactor was set to 3 L. *Streptomyces albopictus* fermentation seed culture obtained in step 2 was inoculated at a 10% inoculum. Fermentation was carried out under initial conditions of pH 6.8, temperature 29°C, and dissolved oxygen 30%, with the pH controlled at 4.0 using ammonia water until fermentation was complete (168 h). At 24 h of fermentation, the pre-prepared fungal mycelium extract with a final concentration of 36 g wet mycelium / L was added to the fermenter in a single batch. When the glucose concentration is below 10 g / L, 600 g / L of glucose solution is added via a peristaltic pump to maintain the concentration in the fermentation broth at 5-15 g / L; when the ammonia nitrogen in the solution is below 0.5 g / L, pre-sterilized 40% (NH4)2SO4 is added externally to maintain the ammonia nitrogen concentration at 0.5-1 g / L, and the final total yield of ε-polylysine is 39.4 g / L.

[0054] Using the optimal fermentation conditions in Example 6, a total ε-polylysine yield of 67.3 g / L was obtained. This means the yield achieved by the method of this invention is 3.0 times that of the traditional ε-polylysine fermentation process, 2.1 times that of the in-situ adsorption-non-co-culture fermentation process with added resin, and 1.7 times that of the process with direct addition of fungal cell extract. Therefore, this fermentation process has a significant product enhancement effect and considerable industrial application value.

[0055] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, such modifications or improvements made without departing from the spirit of the present invention are all within the scope of protection claimed by the present invention.

Claims

1. A method for synthesizing ε-polylysine through streptomycete-mold co-culture, characterized in that: Streptomyces leucovorum IFO14147 was used as the ε-polylysine producing strain, and Aspergillus niger was used as the inducing strain. The two strains were co-cultured under the same culture medium and fermentation conditions to synthesize ε-polylysine. During the co-culture process, the fermentation broth was immersed in cation exchange resin to control the concentration of ε-polylysine in the system. By utilizing the inhibitory effect of ε-polylysine on mold, an appropriate concentration of ε-polylysine was selected to control mold growth. In this way, the mold continuously synthesized a biological inducer that could promote the synthesis of ε-polylysine by Streptomyces leucovorum, ultimately resulting in an increase in fermentation yield. The cation exchange resin includes Amberlite IRC-50, HD-2, D004, or D152; Includes the following steps: Step 1: Activation of microbial strains Spread the spores of Streptomyces albopictus on solid betanna medium and incubate them at 30°C for 8-10 days until the spores mature. Spread Aspergillus niger spores on PDA medium and incubate at 30°C for 8-10 days until the spores mature. Step 2: Fermentation of Seed Culture Scrape 2-3 loops of Streptomyces spores and inoculate them into a 500 mL Erlenmeyer flask containing 60 mL of M3G medium. Incubate at 30°C and 200 rpm for 1-2 days in a constant temperature shaking incubator. Scrape 2-3 rings of Aspergillus niger spores and inoculate them into a 500 mL Erlenmeyer flask containing 80 mL of LM3G medium. Incubate at 30°C and 200 rpm in a constant temperature shaking incubator for 1-3 days. Step 3: Cation exchange resin modification The cation exchange resin was placed in an Erlenmeyer flask, and 1 mol / L ammonia water, 1 mol / L hydrochloric acid, and 1 mol / L ammonia water were added sequentially at 5 times the resin volume. The mixture was shaken on a shaker at 30°C and 200 rpm for 4 hours. After each alkali-acid-alkali modification, the resin was washed multiple times with deionized water until the pH of the washing solution reached 8.

5. The modified cation exchange resin was then wrapped in a mesh cage to obtain a resin package. Step 4: Fermentation process A 5L fermenter was used. M3G culture medium was prepared and sterilized at 121℃ for 20 min. The total liquid volume of the reactor was set to 2-3L. The *Streptomyces cylindrica* fermentation seed liquid obtained in step 2 was inoculated for fermentation. From 0h to 60h of fermentation, the *Aspergillus niger* fermentation broth obtained in step 2 was inoculated and the cells were filtered. Thereafter, the pH value was controlled with ammonia water at 3.8-4.2 until the end of fermentation. When the ε-polylysine concentration in the system was higher than 5g / L, the resin immersion operation was started to adsorb the excess ε-polylysine and maintain the ε-polylysine concentration in the fermenter at 4-7g / L. In step 4, the inoculum size of the *Streptomyces simonii* fermentation seed liquid is 6-10%. In step 4, the inoculum size of the Aspergillus niger fermentation broth filtered is 10%-25%.

2. The method for synthesizing ε-polylysine through streptomyces-mold co-culture according to claim 1, characterized in that: In step 4, the fermentation conditions after inoculating the Streptomyces simulans seed culture are: initial pH 6.0-7.5, temperature 25-33℃, and dissolved oxygen 20%-40%.

3. The method for synthesizing ε-polylysine through streptomyces-mold co-culture according to claim 1, characterized in that: Different mold fermentation broths obtained in step 2 were inoculated with filtered bacterial cells after 48 hours of fermentation.

4. The method for synthesizing ε-polylysine through streptomyces-mold co-culture according to claim 1, characterized in that: During fermentation, when the glucose concentration in the system is below 10 g / L, 600 g / L of glucose solution is added via a peristaltic pump to maintain the concentration in the fermentation broth at 5-15 g / L; when the ammonia nitrogen concentration in the system is below 0.5 g / L, pre-sterilized 40% (NH4)2SO4 is added externally to maintain the ammonia nitrogen concentration at 0.5-1.0 g / L.