Kitchen waste pretreatment method for propionic acid fermentation as well as special fungicide and fermentation aid

Abstract

The invention belongs to the field of bioengineering and solid waste resource utilization, and relates to a kitchen waste pretreatment method for propionic acid fermentation, a special microbial inoculum and a fermentation assistant.The kitchen waste pretreatment method for propionic acid fermentation comprises the steps that kitchen waste is crushed into particles of 2-5 mm, the particles are mixed with water according to the solid content of 40%-80%, the mixture is heated at the temperature of 50-80 DEG C and then filtered, and a mixture is obtained; filtering to obtain filtrate; performing oil-water separation on the filtrate to obtain a water phase and an oil phase; adding 1%-10% (v / v) of the obtained oil phase back into the water phase, and emulsifying at the rotating speed of 800-1000rpm; sequentially carrying out enzymolysis treatment of lipase hydrolysis, alpha-high-temperature amylase hydrolysis and glucoamylase hydrolysis on the emulsified treatment liquid; after impurity adsorption, enzyme deactivation is performed to obtain the propionibacterium fermentation substrate. The strain can be used for efficient substrate conversion, has convenient microbial inoculum application, optimizes the fermentation process, improves economic and environmental benefits, and has industrial application potential.

Description

Technical Field

[0001] This invention relates to the fields of bioengineering and solid waste resource utilization, and particularly to a pretreatment method for kitchen waste for propionic acid fermentation, as well as a special microbial agent and fermentation aid. Background Technology

[0002] Propionic acid is an important short-chain fatty acid widely used in the production of food preservatives, feed additives, chemical intermediates, and biodegradable plastics (such as polyhydroxyalkanoates, PHA). Currently, industrial production of propionic acid mainly relies on chemical synthesis methods (such as the Rapper process) and microbial fermentation. Microbial fermentation has attracted much attention due to its environmental friendliness and mild conditions; however, its mainstream process still uses grain-based raw materials (such as glucose and glycerol) as fermentation substrates, which presents problems such as high raw material costs and competition with human food and animal feed, limiting its large-scale application.

[0003] Meanwhile, the global production of food waste is enormous, rich in organic matter such as starch, cellulose, protein, and oil. Traditional food waste treatment methods, such as landfill and incineration, easily generate pollutants such as leachate, greenhouse gases, and dioxins, causing serious environmental problems and resource waste. Therefore, converting food waste into high-value-added chemicals is an important direction for achieving its resource recovery, volume reduction, and harmless treatment.

[0004] In existing technologies, studies have attempted to utilize food waste for anaerobic fermentation to produce volatile fatty acids (VFAs), including propionic acid. A typical existing technical approach involves: crushing the food waste; enzymatic hydrolysis using cellulase and hemicellulase; further treatment using hot alkali; and finally inoculating with Propionibacterium acnes (such as CGMCC1.2232) for propionic acid fermentation.

[0005] However, this existing technical solution has the following significant drawbacks: 1. Poor substrate adaptability and low fermentation efficiency: Food waste has a complex composition. If only cellulase and hemicellulase are used for treatment, the saccharification efficiency is insufficient for food waste rich in starch and oil, resulting in a low concentration of usable carbon sources (especially glucose) in the fermentation substrate. Simultaneously, the treated substrate may have insufficient amino acid content and may contain proteins that are difficult to ferment and excessive oil content. These factors are all unfavorable for the initial growth and proliferation of Propionibacterium acnes, leading to slow fermentation start-up, long cycle, and low final propionic acid yield. High oil content can also cause emulsification and clogging problems during subsequent fermentation and product extraction, increasing the difficulty and cost of separation and purification.

[0006] 2. Inconvenience in strain preservation and use: During fermentation, Propionibacterium produces acid, which rapidly acidifies the culture medium, hindering bacterial growth and reducing the activity and stability of the seed culture, resulting in a short shelf life. In industrial production, obtaining sufficient live bacteria requires cumbersome multi-stage propagation, which is complex, time-consuming, and labor-intensive. For small- to medium-scale or distributed processing scenarios (such as communities and restaurants), this complex strain preparation process is difficult to implement, limiting the widespread application of the technology.

[0007] 3. Insufficient control over the fermentation process, resulting in yields below industrial-scale levels: Current technology lacks specialized nutrient fortifiers (fermentation aids) tailored to the characteristics of food waste hydrolysate. While food waste hydrolysate contains carbon sources, it often lacks key nitrogen sources, vitamins, and trace elements required for efficient propionic acid synthesis by Propionibacterium. Without these supplements, fermentation intensity is weak, and the final propionic acid concentration and conversion rate are difficult to meet the economic requirements for industrial-scale production.

[0008] Therefore, there is an urgent need in this field to develop an integrated technology solution that can overcome the above-mentioned defects and achieve efficient, stable, and convenient conversion of food waste into propionic acid. Summary of the Invention

[0009] To address the aforementioned technical problems, this invention provides a method for pretreating kitchen waste for propionic acid fermentation, characterized by comprising the following steps: S11, crush kitchen waste into 2-5mm particles, mix with water at a solid content of 40%-80%, heat at 50-80℃ and then filter to obtain filtrate; S12, the filtrate is subjected to oil-water separation to obtain an aqueous phase and an oil phase; S13, add 1% to 10% of the oil phase obtained in step S12 back to the aqueous phase and emulsify at a speed of 800 to 1000 rpm; S14, the emulsified solution is subjected to a three-step enzymatic hydrolysis process: lipase hydrolysis, α-high temperature amylase hydrolysis, and glucosyl amylase hydrolysis. S15, the hydrolysate after the three-step enzymatic hydrolysis is subjected to a second oil removal treatment, and the free oil precipitated during the hydrolysis process is removed by centrifugation. S16, the hydrolysate after secondary degreasing is passed through an adsorption column packed with macroporous adsorption resin to adsorb and remove pigments, residual oil decomposition products and possible fermentation inhibitors. S17, after being adsorbed by impurities, has its enzymes inactivated to obtain the Propionibacterium fermentation substrate.

[0010] Preferably, the specific conditions for the three-step enzymatic hydrolysis treatment are as follows: For lipase hydrolysis, the lipase addition amount is 200-400 U / L, pH is 6.6-7.0, temperature is 30-60℃, and time is 2-16 hours; Hydrolysis with α-thermoamylase: α-thermoamylase addition amount 200-800 U / L, temperature 70-100℃, time 5-24 hours; Hydrolysis with glucoamylase: glucoamylase addition amount 300-500 U / L, temperature 40-60℃, time 12-24 hours.

[0011] On the other hand, the present invention provides a direct-inoculation Propionibacterium inoculum for propionic acid fermentation, which is prepared by freeze-drying a mixture of inoculum sludge and a protectant, wherein the protectant comprises the following components dissolved in a 0.1M phosphate buffer solution at pH 7.00: Acid-hydrolyzed casein 1–5 g / 100 mL; Lactose 3-7g / 100mL; Trehalose 3-7g / 100mL; Monosodium glutamate 0.5–3 g / 100 mL; Glycine 0.05–0.3 g / 100 mL; Sodium ascorbate 0.05-0.3g / 100mL; wherein the mass ratio of the bacterial sludge to the protective agent is 1:2 to 1:3.

[0012] Preferably, the freeze-drying conditions are: quick-freezing at -80°C for 4 to 9 hours, then freeze-drying at -25°C to -20°C under vacuum for 18 to 26 hours, and then slowly heating to 15°C to 22°C until the moisture content is 3% to 5%.

[0013] Preferably, the viable count of the bacterial agent is not less than 1×10⁻⁶. 11 CFU / g.

[0014] On the other hand, the present invention provides a fermentation aid for propionic acid fermentation, comprising the following components in parts by weight: 450-550 parts peptone; 450-550 parts of yeast extract; 80-120 parts sugarcane molasses; 250-350 parts of dried corn starch powder; 130-170 parts of dipotassium hydrogen phosphate; 15-19 parts of ferrous chloride; 22-28 parts magnesium sulfate; 1 to 3 parts cobalt chloride.

[0015] On the other hand, the present invention provides a method for propionic acid fermentation using kitchen waste, comprising the following steps: S21, using the above method to treat kitchen waste, a Propionibacterium fermentation substrate is obtained; S22, dilute the fermentation substrate obtained in step S21 to a glucose concentration of 18-22 g / L, then add the above-mentioned fermentation aid at a concentration of 15-30 g / L, and sterilize to obtain the initial fermentation medium. S23, after resuspending the above-mentioned direct-inoculation Propionibacterium inoculum in sterile water, inoculate it into the initial fermentation medium at a dosage of 10-30g inoculum / L initial fermentation medium; S24 was subjected to anaerobic fermentation at pH 6.7–6.9 and temperature 30–34℃, with a high concentration of glucose solution added as feed during the fermentation process until fermentation was completed. S25. After fermentation, the fermentation broth is subjected to solid-liquid separation to remove microbial cells and other insoluble solid waste. S26, purify the supernatant containing propionic acid obtained by separation, the purification includes: acidifying the supernatant to pH 2.0-3.0, then extracting the propionic acid therein with an organic solvent, and finally obtaining a propionic acid product with a purity ≥99.0% by distillation.

[0016] Preferably, in step S24, the concentration of the high-concentration glucose solution is 600–800 g / L.

[0017] Preferably, in step S24, the fermentation cycle is 240 to 260 hours.

[0018] On the other hand, the present invention provides the use of propionic acid produced by the above method in the preparation of food preservatives, feed additives or biodegradable plastic polyhydroxy fatty acid esters.

[0019] Compared with the prior art, the present invention has the following main advantages: By employing a pretreatment process of crushing, filtering, oil-water separation, and three-step enzymatic hydrolysis (lipase / α-amylase / glucosylamylase), the starch and oil in kitchen waste are efficiently converted into glucose, solving the problems of incomplete saccharification and oil interference in traditional methods. The glucose concentration of the fermentation substrate after treatment can reach 30-50 g / L, laying a solid foundation for high propionic acid production. By adopting an innovative direct-inoculation microbial agent preparation method and optimizing the protective agent formula and freeze-drying process, the microbial agent has a high viable count, good stability, and long shelf life. It can be directly added to the fermentation tank without complicated expansion steps, which greatly simplifies the operation process and lowers the technical threshold. It is particularly suitable for distributed, small-scale on-site treatment of kitchen waste. (3) In view of the unbalanced nutrient composition of kitchen waste hydrolysate, a special fermentation aid was designed to supplement nitrogen source, vitamin, trace elements and buffer salt, creating the best environment for the growth of Propionibacterium and acid production. Combined with the fed-feed fermentation strategy, the final concentration and conversion rate of propionic acid were significantly improved. (4) Replacing food raw materials with cheap kitchen waste significantly reduces production costs. At the same time, it realizes the high-value utilization of organic waste, reduces environmental pollution, and meets the requirements of green, circular and sustainable development. (5) Data from the examples show that, with a 50L fermenter, the propionic acid yield can reach 60.22g / L and the conversion rate can reach 60.17%, which is at the level of industrial production, proving the feasibility and superiority of this technical solution. Attached Figure Description

[0020] To more clearly illustrate the solutions in this invention, the accompanying drawings used in the description of the embodiments of this invention will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0021] Figure 1 This is a flowchart of the pretreatment method for kitchen waste for propionic acid fermentation according to the present invention. Detailed Implementation

[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein in the specification is for the purpose of describing particular embodiments only and is not intended to limit the invention; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings are used to distinguish different objects and not to describe a particular order.

[0023] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of the invention. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0024] To enable those skilled in the art to better understand the present invention, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings.

[0025] Example 1: Pretreatment of kitchen waste and preparation of fermentation substrate Figure 1 This is a flowchart of the pretreatment method for food waste used in propionic acid fermentation according to the present invention. Figure 1As shown, the method for pretreatment of kitchen waste for propionic acid fermentation includes the following steps: S11, crush kitchen waste into 2-5mm particles, mix with water at a solid content of 40%-80%, heat at 50-80℃ and then filter to obtain filtrate; S12, the filtrate is subjected to oil-water separation to obtain an aqueous phase and an oil phase; S13, add 1% to 10% of the oil phase obtained in step S12 back to the aqueous phase and emulsify at a speed of 800 to 1000 rpm; S14, the emulsified solution is subjected to a three-step enzymatic hydrolysis process: lipase hydrolysis, α-high temperature amylase hydrolysis, and glucosyl amylase hydrolysis. S15, the hydrolysate after the three-step enzymatic hydrolysis is subjected to a second oil removal treatment, and the free oil precipitated during the hydrolysis process is removed by centrifugation. S16, the hydrolysate after secondary degreasing is passed through an adsorption column packed with macroporous adsorption resin to adsorb and remove pigments, residual oil decomposition products and possible fermentation inhibitors. S17, after being adsorbed by impurities, has its enzymes inactivated to obtain the Propionibacterium fermentation substrate.

[0026] In this embodiment, canteen kitchen waste (mainly rice, noodles, vegetables, and a small amount of meat grease) is first collected. It is then crushed into particles approximately 3mm using a crusher. 10kg of the crushed waste is weighed and mixed with 15kg of water (containing approximately 40% solids). The mixture is heated in a 70℃ water bath for 30 minutes, then filtered through a 100-mesh sieve, discarding the solid residue. The filtrate is then separated into oil and water using a three-phase separator. 5% of the separated oil (oil phase) is added back to the aqueous phase and emulsified at 1000rpm for 15 minutes. Subsequently, a three-step enzymatic hydrolysis is performed: Lipase hydrolysis was performed by adding 300 U / L of commercial lipase (Novozymes) to the emulsion, adjusting the pH to 6.8 with 1M NaOH, and hydrolyzing at 45°C for 8 hours.

[0027] High-temperature amylase hydrolysis was performed by raising the temperature of the hydrolysate to 90°C, adding 500 U / L of α-high-temperature amylase (Genetronics), and hydrolyzing for 12 hours.

[0028] Perform saccharifying enzyme hydrolysis, cool the hydrolysate to 50°C, add 400 U / L of glucosyl amylase (Genentech), and hydrolyze for 18 hours.

[0029] A second degreasing step was performed. The enzymatically hydrolyzed solution was centrifuged at 4°C and 8000 rpm for 15 minutes. A distinct lipid layer was observed to precipitate on the surface. The free lipids on top were carefully removed using a pipette. This step reduced the residual lipid content in the hydrolysate from approximately 1.5% to below 0.3%.

[0030] Next, impurity adsorption is performed. The hydrolysate after the second oil removal is pumped into a glass adsorption column packed with AB-8 macroporous adsorption resin (column height to diameter ratio of 10:1) at a flow rate of 2 column volumes (BV) / hour. The outflowing liquid is collected.

[0031] Effect comparison: In terms of sensory aspects: After adsorption treatment, the color of the hydrolysate changed from dark brown to light yellow, and the transparency was significantly improved.

[0032] Indicator detection: The total phenol content of the hydrolysate before and after treatment (Folin-Ciocalteu method) and the BOD5 / COD ratio used to assess the level of fermentation inhibitors were detected.

[0033] The total phenol content decreased from 850 mg / L before treatment to 210 mg / L.

[0034] The BOD5 / COD ratio increased from 0.45 to 0.62, indicating enhanced biodegradability and reduced inhibitors.

[0035] After impurity adsorption, the enzymes were inactivated by heating at 95℃ for 15 minutes. Sampling and analysis revealed that the glucose concentration of the resulting Propionibacterium fermentation substrate was 42.5 g / L.

[0036] Fermentation validation: Small-scale fermentation experiments were conducted using hydrolysates that had undergone adsorption treatment and those that had not. The results showed that the experimental group using the adsorbed hydrolysates experienced an approximately 15% increase in propionic acid fermentation rate, and the final propionic acid yield was 8.5% higher than that of the untreated group.

[0037] Example 2: Effect of different adsorption resins on pretreatment effect This embodiment further verifies the effectiveness of passing the hydrolysate after secondary degreasing through an adsorption column packed with macroporous adsorption resin to adsorb and remove pigments, residual oil decomposition products, and possible fermentation inhibitors.

[0038] The method was the same as in Example 1, but the purification effects of three macroporous adsorption resins with different polarities (AB-8, X-5, and D101) on the hydrolysate were compared. The results are as follows: AB-8 resin (weakly polar): It has the best effect on removing pigments and phenols, produces the lightest liquid color after treatment, has the highest increase in BOD5 / COD ratio, and has the best subsequent fermentation effect.

[0039] X-5 resin (non-polar): It has a strong adsorption capacity for oil residues, but its decolorization effect is slightly inferior to AB-8.

[0040] D101 resin (non-polar): Its overall performance is between the two. Conclusion: AB-8 resin is the preferred adsorption medium in this pretreatment system.

[0041] Example 3: Pretreatment effect of kitchen waste from different sources Two types of kitchen waste, one from restaurants (high in fat and protein) and the other from households (high in starch and low in fat), were used as raw materials and pretreated according to the method described in Example 1. The results are as follows: The glucose concentration of the fermentation substrate after restaurant waste treatment was 35.2 g / L.

[0042] The glucose concentration of the fermentation substrate after household waste treatment was 48.7 g / L. The results indicate that the method of this invention has good adaptability to kitchen waste of different compositions and can achieve high glucose concentrations in all cases.

[0043] Example 4: Preparation of bacterial agent protectant buffer solution Accurately weigh 1.36 g of potassium dihydrogen phosphate and 1.74 g of dipotassium hydrogen phosphate, dissolve them in 100 mL of ultrapure water, stir to dissolve, and calibrate to pH=7.00 using a pH meter to obtain 0.1 M phosphate buffer solution, which is stored at 4 °C for later use.

[0044] Example 5: Preparation of bacterial agent protectant solution Take 100 mL of the phosphate buffer prepared in Example 3, and add 3 g of acid-hydrolyzed casein, 5 g of lactose, 5 g of trehalose, 1.5 g of sodium glutamate, 0.15 g of glycine, and 0.15 g of sodium ascorbate in sequence. Stir magnetically until completely dissolved, and store the solution in an ice-water bath (0-4°C) for later use.

[0045] Example 6: Preparation of Propionibacterium seed culture Propionibacterium freudenreichii CGMCC 1.2232 was streaked onto seed culture plates (containing 10 g / L yeast extract, 10 g / L peptone, 1 g / L potassium dihydrogen phosphate, 1 g / L dipotassium hydrogen phosphate, pH 7.0) and anaerobically cultured at 32°C for 72 hours. A single colony was picked and inoculated into an Erlenmeyer flask containing 50 mL of the same liquid seed culture medium, and statically anaerobically cultured at 32°C for 48 hours as the primary seed culture. This 5% inoculum was then transferred to a fermenter containing 1 L of seed culture medium. The temperature was controlled at 32°C and pH 6.8 (controlled by automatic ammonia addition), and fermentation was carried out for 60 hours. Fermentation was stopped when the OD600 value reached 70. The entire process was conducted under aseptic conditions.

[0046] Example 7: Preparation of Direct-Inoculation Propionibacterium Inoculum The seed fermentation broth prepared in Example 6 was aseptically filtered to collect wet bacterial sludge. 10g of wet bacterial sludge was weighed and mixed thoroughly with 20g of the preservative solution prepared in Example 4 in a sterile container to form a bacterial suspension. In an anaerobic workstation, the bacterial suspension was dispensed into sterile vials, each filled to approximately 1 / 3 of its volume. The vials were then rapidly transferred to an ultra-low temperature freezer at -80°C and frozen for 6 hours. The frozen sample was then placed in a freeze dryer pre-cooled to -40°C, and the vacuum pump was turned on for freeze-drying at -22°C for 22 hours. The temperature was then slowly increased to 18°C, and drying continued until the sample moisture content was 4.2%. Sampling and testing revealed a viable bacterial count of 3.5 × 10⁻⁶. 11 CFU / g.

[0047] Example 8: Preparation of Fermentation Aid Weigh the following components: 500g peptone, 500g yeast extract, 100g sugarcane molasses, 300g corn syrup powder, 150g dipotassium hydrogen phosphate, 17g ferrous chloride, 25g magnesium sulfate, and 2g cobalt chloride. Place all solid powders in a mixer and mix at low speed for 30 minutes until homogeneous. Seal the package and store in a cool, dry place.

[0048] Example 9: Propionic acid fermentation experiment in a 50L fermenter The Propionibacterium fermentation substrate prepared in Example 1 was diluted with water to a glucose concentration of 20 g / L. 30 L of this diluted solution was taken, and 750 g of the fermentation aid prepared in Example 7 was added. After stirring to dissolve, the solution was sterilized at 115°C for 30 minutes. After cooling, it was transferred to a 50 L fermenter as the initial fermentation medium. 20 g of the inoculum prepared in Example 6 was resuspended in 100 mL of sterile water and added entirely to the fermenter. Fermentation conditions were controlled as follows: temperature 32°C, pH 6.8 (controlled by adding 10 M NaOH), and rotation speed 200 rpm (for mixing only, not strictly aerobic). Starting from the 48th hour of fermentation, a 700 g / L glucose solution was added as a feedstock, with the feed rate controlled to maintain a certain metabolic activity. Fermentation lasted for 240 hours.

[0049] Experimental results: Final propionic acid yield: 60.22 g / L; Conversion rate of glucose to propionic acid: 60.17%; Fermentation cycle: 240 hours.

[0050] Example 10: Comparison of fermentation effects with different amounts of microbial agents Except for changing the amount of bacterial agent added, the other conditions are the same as in Example 9.

[0051] Experimental group 1: Microbial agent addition amount 10g / L. The final propionic acid yield was 55.18g / L, and the conversion rate was 55.10%.

[0052] Experimental Group 2: Microbial agent addition amount 20 g / L (same as Example 9). The final propionic acid yield was 60.22 g / L, with a conversion rate of 60.17%.

[0053] Experimental Group 3: Inoculum dosage 30 g / L. Final propionic acid yield was 61.05 g / L, conversion rate 61.00%. The results show that increasing the inoculum dosage can improve fermentation rate and yield, but considering cost, 20 g / L is the optimal choice.

[0054] Example 11: Comparison of fermentation effects with different amounts of fermentation aids Except for changing the amount of fermentation aid added, the other conditions are the same as in Example 9.

[0055] Experimental group 1: Fermentation aid dosage 15 g / L. Final propionic acid yield was 52.41 g / L.

[0056] Experimental group 2: Fermentation aid dosage 22.5 g / L. Final propionic acid yield was 58.90 g / L.

[0057] Experimental group 3: Fermentation aid dosage 30 g / L. Final propionic acid yield was 60.50 g / L.

[0058] The results showed that fermentation aids were crucial for increasing yield, with an effective range of 22.5–30 g / L.

[0059] Example 12: Stability Test of Microbial Agent Storage The bacterial agent prepared in Example 6 was dispensed and stored at 4°C and room temperature (25°C), respectively. Viable bacterial counts were measured on days 0, 30, 90, and 180 after storage.

[0060] result: Storage at 4℃: viable bacterial count after 0 days is 3.5 × 10⁻⁶. 11 CFU / g; 3.2 × 10⁻⁶ after 30 days. 11 CFU / g; 2.8 × 10⁻⁶ after 90 days. 11 CFU / g; after 180 days, it was 2.1×10⁻⁶. 11 CFU / g.

[0061] Room temperature storage: viable count on day 0 is 3.5 × 10⁻⁶. 11 CFU / g; 2.0×10 after 30 days. 11 CFU / g; 8.5×10 after 90 days. 10 CFU / g; 3.2 × 10⁻¹⁰ after 180 days. 10 CFU / g. The results showed that the bacterial agent of the present invention maintained a high number of viable bacteria after being stored at 4°C for 6 months, demonstrating good stability.

[0062] Example 13: Post-processing of fermentation products (waste removal and solvent extraction purification) Take 5L of the fermentation mash after the fermentation of Example 9 is completed.

[0063] Solid-liquid separation: First, the mash is coarsely filtered using a plate and frame filter press to remove most of the bacteria and solid particles. Then, the coarse filtrate is finely filtered through a 0.22 μm ceramic membrane filtration system to obtain a clear and transparent liquid containing propionic acid.

[0064] Solvent extraction: The pH of the clarified fermentation broth was adjusted to 2.5 with concentrated sulfuric acid. Then it was mixed with an equal volume of methyl isobutyl ketone (MIBK) and extracted in an extraction vessel at 50°C with stirring for 30 minutes. After standing and separation, the organic phase rich in propionic acid was collected.

[0065] Preliminary results: Propionic acid recovery rate reached over 85% after a single extraction. High-purity propionic acid could be further obtained through back-extraction and distillation. This step demonstrates the feasibility of effectively separating and initially concentrating propionic acid from complex fermentation mash.

[0066] Example 14: Obtaining high-purity propionic acid through a complete post-processing procedure Take 30L of fermentation broth from Example 9 after fermentation is completed, and perform solid-liquid separation using the method described in Example 13 to obtain a clarified fermentation broth.

[0067] Acidification and extraction: The clarified liquid was acidified to pH 2.5 with sulfuric acid and then extracted with MIBK using a three-stage countercurrent extraction method.

[0068] Back-extraction: Combine all organic phases, use deionized water as the back-extraction agent, and perform back-extraction at pH 10.0 (adjusted with NaOH) to back-extract propionic acid from the organic phase to the aqueous phase.

[0069] Distillation: The aqueous phase rich in propionic acid is fed into a distillation column. Most of the water is first evaporated at atmospheric pressure, and then distillation is carried out under reduced pressure to collect the propionic acid fraction with the required boiling range.

[0070] Final product: 2.1 kg of colorless and transparent propionic acid was obtained. High-performance liquid chromatography (HPLC) analysis showed that the propionic acid purity reached 99.3%, and all indicators met the standards for food-grade propionic acid. This example fully demonstrates the entire process from food waste fermentation mash to high-value-added chemical products.

[0071] Example 15: Comparative Experiment with Traditional Methods (Comparative Example) The process and fermentation are carried out according to the existing technical solutions described in the background art: the same batch of kitchen waste is only crushed, treated with cellulase and hemicellulase, then treated with hot alkali, and finally inoculated with Propionibacterium acnes (same strain) that has been expanded by traditional liquid seed culture for fermentation, without adding the fermentation aid of the present invention.

[0072] result: The concentration of glucose in the substrate after treatment was 15.8 g / L.

[0073] Final propionic acid yield: 28.45 g / L.

[0074] Conversion rate: 45.0% (based on available sugar).

[0075] Fermentation cycle: 48 hours of seed culture is required first, and the total cycle is as long as 288 hours.

[0076] Compared with Example 9, the propionic acid yield and conversion rate of the present invention are significantly higher than those of the comparative example, and the microbial agent is convenient to use and the total operation time is shorter.

[0077] Obviously, the embodiments described above are merely some embodiments of the present invention, not all embodiments. The accompanying drawings show preferred embodiments of the present invention, but do not limit the patent scope of the present invention. The present invention can be implemented in many different forms; rather, these embodiments are provided to provide a more thorough and complete understanding of the disclosure of the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing specific embodiments, or make equivalent substitutions for some of the technical features. Any equivalent structures made using the content of this specification and drawings, directly or indirectly applied to other related technical fields, are similarly within the patent protection scope of this invention.

Claims

1. A kitchen waste pretreatment method for propionic acid fermentation, characterized by, It comprises the following steps: S11, crushing the kitchen waste into 2-5mm particles, mixing with water at a solid content of 40%-80%, heating at 50-80℃ and then filtering to obtain filtrate; S12, separating the oil and water of the filtrate to obtain water phase and oil phase; S13, adding 1%-10% of the oil phase obtained in step S12 to the water phase and emulsifying at a speed of 800-1000rpm; S14, sequentially performing three-step enzymatic hydrolysis of lipase hydrolysis, α-high temperature amylase hydrolysis and glucoamylase hydrolysis on the emulsified treatment liquid; S15, performing secondary deoiling treatment on the hydrolysis liquid after three-step enzymatic hydrolysis to remove free oil and fat precipitated in the hydrolysis process by centrifugal separation; S16, passing the hydrolysis liquid after secondary deoiling through an adsorption column filled with macroporous adsorption resin to remove pigments, residual oil and fat decomposition products and possible fermentation inhibitors by adsorption; S17, deactivating the enzyme after impurity adsorption to obtain propionic acid bacteria fermentation substrate.

2. The method of claim 1, wherein, The specific conditions of the three-step enzymatic hydrolysis are as follows: For lipase hydrolysis, the lipase addition amount is 200-400U / L, the pH is 6.6-7.0, the temperature is 30-60℃, and the time is 2-16 hours; For α-high temperature amylase hydrolysis, the α-high temperature amylase addition amount is 200-800U / L, the temperature is 70-100℃, and the time is 5-24 hours; For glucoamylase hydrolysis, the glucoamylase addition amount is 300-500U / L, the temperature is 40-60℃, and the time is 12-24 hours.

3. A direct vat propionic acid bacteria agent for propionic acid fermentation, characterized by, It is prepared by mixing the bacterial slurry and the protective agent, and then freeze-drying, wherein the protective agent comprises the following components dissolved in 0.1M phosphate buffer at pH 7.00: Acid-hydrolyzed casein 1-5g / 100mL; Lactose 3-7g / 100mL; Trehalose 3-7g / 100mL; Sodium glutamate 0.5-3g / 100mL; Glycine 0.05-0.3g / 100mL; Sodium ascorbate 0.05-0.3g / 100mL; wherein the mass mixing ratio of the bacterial slurry to the protective agent is 1:2 to 1:

3.

4. The bacterial agent of claim 3, characterized in that, The freeze-drying conditions are as follows: quick freezing at -80℃ for 4-9 hours, then freeze-drying at -25℃ to -20℃ under vacuum conditions for 18-26 hours, and then slowly warming up to 15℃-22℃ until the water content is 3%-5%.

5. The bacterial agent of claim 3 or 4, characterized in that, The viable cell count of the bacterial agent is not less than 1 x 10 11 CFU / g.

6. A fermentation aid for propionic acid fermentation, characterized in that, It is composed of the following components by weight: Peptone 450-550 parts; Yeast extract powder 450-550 parts; Cane molasses 80-120 parts; Corn dry pulp powder 250-350 parts; Dipotassium hydrogen phosphate 130-170 parts; Ferrous chloride 15-19 parts; Magnesium sulfate 22-28 parts; Cobalt chloride 1-3 parts.

7. A method for producing propionic acid fermentation using food waste, characterized by, It comprises the following steps: S21, treating the kitchen waste by the method of claim 1 or 2 to obtain propionic acid bacteria fermentation substrate; S22, diluting the fermentation substrate obtained in step S21 to a glucose concentration of 18-22g / L, then adding the fermentation aid of claim 6 at a concentration of 15-30g / L, and sterilizing to obtain an initial fermentation medium; S23, resuspend the Propionibacterium sp. inoculum of any one of claims 3-5 with sterile water, and then add the resuspended Propionibacterium sp. inoculum into the initial fermentation medium at a dosage of 10-30 g inoculum / L of the initial fermentation medium; S24, perform anaerobic fermentation at a pH of 6.7-6.9 and a temperature of 30-34°C, and perform feeding by adding high-concentration glucose solution during the fermentation until the fermentation is completed; S25, after the fermentation is completed, perform solid-liquid separation on the fermented broth to remove the bacterial cells and other insoluble solid waste; S26, purify the supernatant containing propionic acid obtained by the separation, and the purification comprises: acidifying the supernatant to a pH of 2.0-3.0, then extracting propionic acid from the supernatant using an organic solvent, and finally obtaining propionic acid products with a purity of ≥99.0% through rectification.

8. The method of claim 7, wherein, In step S24, the concentration of the high-concentration glucose solution is 600-800 g / L.

9. The method of claim 7, wherein, In step S24, the fermentation period is 240-260 hours.

10. Use of propionic acid produced by the method of any one of claims 7-9 in the preparation of food preservatives, feed additives, or biodegradable plastics polyhydroxyalkanoates.

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