Extraction process of a microalgae nannochloropsis active extract against bacteria and candida albicans

Abstract

The present application belongs to the technical field of natural product functionalization, and particularly relates to an extraction process of a micro-micro green algae active extract against bacteria and Candida albicans. In order to overcome the high energy consumption of microalgae harvesting, low extraction rate of active ingredients, and narrow antibacterial spectrum of the extract, the present application first adopts an alkaline environment assisted electroflocculation harvesting combined with a low-temperature centrifugal dewatering pretreatment method to harvest microalgae, then adopts an ultrasonic cell disruption-enzyme hydrolysis decontamination synergistic algal powder extraction pretreatment strategy and an ethanol and alkali co-extraction-precipitation-lyophilization preservation composite extraction strategy to extract the microalgae antibacterial active ingredients. The micro-micro green algae active extract obtained has high inhibition activity against gram-negative / positive bacteria and Candida albicans, not only overcomes the defect that traditional natural antibacterial agents have weak antifungal effect, but also realizes green and efficient preparation of natural antibacterial agents.

Description

Technical Field

[0001] This invention belongs to the field of natural product functionalization technology, specifically relating to an extraction process for an active extract of *Micrococcus pluvialis* that is antibacterial and effective against *Candida albicans*. Background Technology

[0002] Pathogenic microorganisms refer to microorganisms that can invade the human body and cause infection or infectious diseases. Among them, bacteria and fungi, due to their wide distribution in the environment and potential hazards, have always been a focus of attention in the safety and efficacy evaluation of daily chemical products, and national standards have also made clear limits on their levels. Daily chemical products are closely related to the human living environment, and microbial contamination or proliferation can not only lead to product deterioration but also pose a threat to the health of users. Therefore, improving the antibacterial ability of daily chemical products is crucial. Among the pathogenic or opportunistic pathogenic microorganisms related to the efficacy and safety of daily chemical products, typical examples include Gram-negative bacteria such as Escherichia coli, Gram-positive bacteria such as Staphylococcus aureus, yeasts such as Candida albicans, and opportunistic pathogens such as Pseudomonas aeruginosa. It is worth noting that the volatile compounds produced by the metabolism of Staphylococcus aureus and Pseudomonas aeruginosa are the main sources of odor. Therefore, these two strains are also the core targets of the deodorizing function of daily chemical products and are collectively referred to as odor-producing bacteria. Given the widespread presence of these microorganisms in the environment, the industry's demand for natural, safe alternatives with broad-spectrum antibacterial activity is increasingly urgent. Developing antibacterial daily chemical products that combine broad-spectrum activity and high efficiency has become a significant industry trend. Microalgae, as a renewable biomass resource, can synthesize a variety of secondary metabolites with antibacterial activity, making them an ideal alternative to traditional chemical antibacterial agents. These microalgae-derived active ingredients not only exhibit excellent inhibitory potential against the aforementioned pathogenic and odor-producing bacteria, but also possess higher biocompatibility compared to chemically synthesized antibacterial agents. This makes the development and application of microalgae active ingredients of significant practical importance in antibacterial research within the daily chemical industry.

[0003] Picochlorum eukaryotum is a marine microalga with potential for developing various bioactive components. However, research on its cultivation, harvesting, and antibacterial components remains largely unexplored. To date, one of the main bottlenecks in the industrial application of microalgae lies in the harvesting and drying of biomass, a stage that typically accounts for nearly half of the total production cost. The core challenge in harvesting microalgae is the low concentration of the algal solution, the tiny size of the cells, their negatively charged surface, and their density being similar to water, leading to significant separation difficulties. Commonly used industrial methods for harvesting microalgae include centrifugation, flocculation sedimentation, flotation, and membrane filtration. However, these methods generally suffer from high energy consumption and limited dehydration efficiency, increasing subsequent drying loads and resulting in high equipment maintenance costs. Furthermore, domestic and international research on microalgae largely focuses on its bioactive components and functions. Although microalgae active substances have been proven to possess antibacterial activity, achieving efficient extraction, purification, and retention of this activity remains a key technological bottleneck restricting its industrial application. Microalgae have tough cell walls, making it difficult for a single solvent to completely penetrate them and fully release all types of active substances. As a result, their extracts often have limited activity and cannot achieve broad-spectrum inhibition against Gram-positive bacteria, Gram-negative bacteria, and fungi simultaneously.

[0004] In summary, given the high energy consumption during microalgae harvesting, the low extraction rate of active ingredients due to the tough cell walls, and the narrow antibacterial spectrum and poor antifungal effect of single-solvent extracts, there is an urgent need to develop an efficient and green preparation process for broad-spectrum antibacterial active ingredients from *Chlorella vulgaris* to obtain natural extracts with efficient and broad-spectrum inhibitory effects against various pathogenic and flavor-producing bacteria. Summary of the Invention

[0005] To overcome the shortcomings of high energy consumption, low extraction rate of active ingredients, and narrow antibacterial spectrum in microalgae harvesting, this invention provides a process for harvesting *Chlorella vulgaris* and preparing its broad-spectrum antibacterial active ingredients. This process comprises two core modules: a high-efficiency, low-energy-consumption microalgae harvesting pretreatment using alkaline electrocoagulation coupled with low-temperature centrifugation; and a composite extraction process using ethanol-alkali co-extraction of broad-spectrum antibacterial active ingredients assisted by ultrasonic cell disruption and enzymatic hydrolysis. This preparation process significantly improves microalgae harvesting efficiency and extraction purity, and the resulting extract exhibits highly effective inhibitory activity against Gram-negative / positive bacteria and *Candida albicans*, particularly enhancing its antifungal effect, thus achieving the green and efficient preparation of natural antibacterial agents.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: The first aspect of this invention provides an extraction process for an active extract of *Pheretima aspergillum*, the process comprising the following steps: S1. Harvesting of *Pheretima asiatica*: *Pheretima asiatica* algal solution was electrocoagulated using an electrocoagulation device. During the electrocoagulation process, the algal solution was maintained in an alkaline environment with a pH of 8.0-9.0. After sedimentation, the concentrated algal solution was collected, washed, and then subjected to solid-liquid separation by low-temperature assisted centrifugation. The resulting wet algal mud was then pre-frozen, freeze-dried, and dried to obtain the final product. The alkaline environment significantly improved the floc formation rate and harvested biomass, while low-temperature assisted centrifugation significantly increased the concentration factor and effectively reduced the water content of the mud, which is beneficial for subsequent drying. Electrocoagulation combined with low-temperature centrifugation under alkaline conditions can produce high-efficiency, low-moisture algal powder raw materials.

[0007] S2. Extraction of active extract of *Chlorella vulgaris*: Add ethanol-alkali mixed solution to *Chlorella vulgaris* obtained in S1, pre-treat with ultrasound, filter and collect the initial extract, treat the microalgae filter residue with protease and DNase, then use the collected initial extract to extract the enzyme-treated microalgae, collect the liquid extract, neutralize with hydrochloric acid and evaporate to dry, then dissolve the organic phase with ethanol, and the resulting liquid is evaporated and lyophilized to obtain the final product.

[0008] Preferably, in S1, the temperature of the low-temperature assisted centrifugation is 0-4℃, the rotation speed is 8000 rpm, and the centrifugation time is 15 min.

[0009] Preferably, in S1, the electrocoagulation time is 20-30 min.

[0010] Preferably, in S1, the electrocoagulation device includes a DC power supply, stainless steel electrode plates, an electrode fixing bracket, and an electrolytic cell. Both the anode and cathode are stainless steel electrode plates, the electrode spacing is 7-15 cm, and the output current of the DC power supply is 1-3 A.

[0011] Preferably, in S2, the ethanol-alkali mixed solution is prepared by mixing 2% NaOH and 75% ethanol in a volume ratio of 1:7-11.

[0012] Preferably, in S2, the ultrasonic pretreatment is performed in a 2-3 second on / 1-2 second off pulse mode, with a power of 400-500W and a time of 8-15 minutes.

[0013] Preferably, in step S2, the enzyme-treated microalgae are extracted with the collected initial extract under stirring conditions, and the extraction is repeated 2-4 times, each time for 1-3 hours.

[0014] The second aspect of the present invention also provides an active extract of *Vibrio hygroscopicus* prepared using the extraction process described in the first aspect.

[0015] The third aspect of the present invention also provides the use of the active extract of *Vibrio hygroscopicus* described in the second aspect in the preparation of antibacterial and / or Candida albicans products.

[0016] Preferably, the product includes antibacterial agents, deodorizing agents, or preservatives, and the bacteria include Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

[0017] Compared with the prior art, the beneficial effects of the present invention are: This invention discloses an extraction process for an active extract of *Pheretima asiatica* that inhibits bacteria and *Candida albicans*. First, microalgae are harvested using an alkaline-assisted electrocoagulation harvesting method combined with low-temperature centrifugation dehydration. Then, a pretreatment strategy of ultrasonic cell disruption and enzymatic hydrolysis for algal powder extraction is employed, along with a composite extraction strategy of ethanol and alkali co-extraction, precipitation, and freeze-drying for preservation to extract the antibacterial active components. The extracted *Pheretima asiatica* active extract exhibits highly efficient inhibitory activity against Gram-negative / positive bacteria and *Candida albicans*.

[0018] Specifically, the present invention has the following advantages: (1) Improved harvesting efficiency and reduced energy consumption: Electrocoagulation in an alkaline environment combined with low-temperature centrifugation enabled high biomass harvesting and low-moisture algal mud preparation, overcoming the bottleneck of microalgae harvesting.

[0019] (2) High extraction efficiency and high purity: Ultrasonic disruption effectively breaks down the tough cell walls of microalgae, improving the release rate of active ingredients; the use of protease and DNase to treat algal residue effectively degrades residual proteins and nucleic acids and other macromolecular impurities, improving the relative purity of subsequent extracts; the use of ethanol-alkali co-extraction method, with ethanol as the extraction medium, separates lipid-soluble and weakly polar components; at the same time, the alkaline environment helps to dissolve and stabilize acidic, alkaline and bound active ingredients in the system, ultimately achieving efficient extraction and enrichment of target active ingredients.

[0020] (3) Broad-spectrum activity, especially significant antifungal effect: The ethanol-alkali co-extract of the present invention has strong inhibitory activity against Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), Gram-positive bacteria (Staphylococcus aureus), and fungi (Candida albicans). In particular, it can completely inhibit Candida albicans even at low concentrations, overcoming the deficiency of traditional natural antibacterial agents with weak antifungal effects. Attached Figure Description

[0021] Figure 1 A comparison of pH changes over time during electrocoagulation harvesting of *Micrococcus pluvialis* under unbuffered (NB), acid-buffered (AB), and alkaline-buffered (BB) conditions. Figure 2 A comparison of the yield (mg) of Micrococcus pluvialis harvested by electrocoagulation over time under unbuffered (NB), acid-buffered (AB), and alkaline-buffered (BB) conditions. Figure 3 A comparison of the floc formation rate of *Micrococcus pluvialis* harvested by electrocoagulation for 10 min under conditions of no buffer (NB), acid buffer (AB), and alkaline buffer (BB); Figure 4 A comparison chart showing the concentration factor of *Micrococcus pluvialis* dehydration pretreatment under three methods: room temperature centrifugation (RT-centrifugation), low temperature centrifugation (LT-centrifugation), and vacuum filtration (Vac-filtration); Figure 5 A comparison chart showing the moisture content (%) of mud pretreated with *Micrococcus pluvialis* under three methods: RT-centrifugation, LT-centrifugation, and Vac-filtration. Figure 6 A comparison chart of the yield (g / g) of *Chlorella vulgaris* extract under five pretreatment methods: no pretreatment (NP), acid pretreatment (AP), alkali pretreatment (BP), microwave pretreatment (MP), and ultrasonic pretreatment (UP). Figure 7 Images of culture dishes and colony counts for the MIC determination of *Escherichia coli* using ethanol extract and ethanol-alkali co-extract; Figure 8 Images of culture dishes and colony counts for the MIC determination of Staphylococcus aureus by ethanol extract and ethanol-alkali co-extract of *Micrococcus pluvialis*. Figure 9 Images of culture dishes and colony counts for the MIC determination of *Candida albicans* using ethanol extract and ethanol-alkali co-extract. Figure 10 The images show the actual culture dish and colony count of the ethanol-alkali co-extract of *Pseudomonas aeruginosa*, a typical flavor-producing bacterium, for MIC determination. Detailed Implementation

[0022] The specific embodiments of the present invention will be further described below. It should be noted that these descriptions are for the purpose of aiding understanding the present invention, but do not constitute a limitation thereof. Furthermore, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

[0023] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods, and the experimental materials used in the following embodiments are all available through conventional commercial channels.

[0024] This invention provides a highly efficient and environmentally friendly process for preparing a microalgae antibacterial agent, which can be used as an antibacterial agent and deodorizer in the daily chemical industry. The preparation process includes a complete workflow from the artificial cultivation and harvesting of *Chlorella vulgaris* cells to the efficient preparation of antibacterial active extracts. It mainly comprises two core steps: first, the microalgae harvesting module employs an alkaline-assisted electrocoagulation harvesting combined with a low-temperature centrifugal dehydration pretreatment method; second, the microalgae antibacterial active ingredient extraction module employs a pretreatment strategy of ultrasonic cell disruption and enzymatic hydrolysis for impurity removal, as well as a composite extraction strategy of ethanol and alkali co-extraction, precipitation, and freeze-drying preservation. This invention aims to improve microalgae harvesting efficiency, extract yield, broad-spectrum antibacterial activity, and antibacterial rate.

[0025] This invention determines the optimal harvesting method by comparing the effects of buffer conditions and dehydration treatment on the harvesting efficiency of *Micrococcus pluvialis* electrocoagulation harvesting. This method enables efficient harvesting of microalgae, reduces the moisture content of the algal sludge, and decreases energy consumption in subsequent drying processes. Simultaneously, by comparing the extract yields of *Micrococcus pluvialis* under different pretreatment methods and the inhibitory effects of traditional ethanol extraction on various experimental bacterial species in the suspension antibacterial effect evaluation, the optimal pretreatment method, extraction process, and their broad-spectrum antibacterial activity are clarified.

[0026] The antibacterial effects of *Escherichia coli* (Gram-negative bacteria), *Staphylococcus aureus* (Gram-positive bacteria), *Candida albicans* (fungus), and *Pseudomonas aeruginosa* (odor-producing bacteria) were evaluated. The results showed that the ethanol-alkali co-extract of *Microalgae* exhibited excellent broad-spectrum antibacterial activity. Compared to the ethanol extract obtained by single solvent extraction, the co-extract showed significantly enhanced antibacterial activity. It not only showed better inhibition rates against *Escherichia coli* and *Staphylococcus aureus* than the traditional ethanol extract, but also achieved excellent inhibition of *Candida albicans*, which showed a lower inhibition rate with the ethanol extract, at a lower effective concentration. This overcomes the weakness of traditional natural antibacterial agents in antifungal activity. The microalgae antibacterial agent prepared by this invention can be used as a natural, broad-spectrum antibacterial raw material in the fields of preservation and deodorization of daily chemical products, and has significant economic and social value.

[0027] To clearly demonstrate the technical solution of the present invention, the following embodiments are arranged in the order of the processes: First, large-scale cultivation of *Micrococcus pluvialis* is carried out (Example 1); then, the optimal microalgae harvesting and solid-liquid separation process is explored and determined to prepare dried algae powder (Example 2); next, the cell wall disruption pretreatment method of the dried algae powder is screened and optimized (Example 3); and based on the obtained optimal pretreatment conditions, the antibacterial active ingredients are deeply extracted (Example 4); finally, the antibacterial effect of the extracted components is evaluated and compared to verify the effect of process optimization on improving the antibacterial effect (Example 5).

[0028] Example 1: Cultivation and recovery of *Vibrio globosum* strain The *Picochlorum eukaryotum* used in this invention was collected from the beach of Tangjiawan, Zhuhai City, Guangdong Province (N 22°20'9'', E 113°35'32''), with the serial number ZH-TW22, and is currently preserved in Room 1003, Haiqin Building 5, Zhuhai Campus, Sun Yat-sen University.

[0029] The cultivation and resuscitation of *Vibrio pulmonale* strains were conducted entirely in a sterile environment free from contamination by other algae. The culture medium was prepared using seawater with a salinity of 20‰. Algal strains stored at 4℃ were inoculated into a 20 L photobioreactor at a volume ratio of 1:10 (algal culture to freshly prepared seawater), with 1 mL / L of F / 2 culture medium stock solution added simultaneously. The cultivation conditions were set as follows: temperature 25℃, light intensity 2000–3000 Lux, using continuous 24-hour illumination. After 7–10 days of cultivation, the algal culture was transferred to a large-volume culture device for scale-up cultivation, until the bacterial OD reached a 10-fold dilution. 680 The value is approximately 0.8.

[0030] Example 2: Harvesting process of *Chlorella vulgaris* We used a combination of electrocoagulation and solid-liquid separation (centrifugation / vacuum filtration) to harvest Microchlorophyll and explore the optimal harvesting method and conditions.

[0031] 2.1 Electrocoagulation Device: The electrocoagulation system consists of a DC power supply (output current 1 A), 304 stainless steel electrode plates, electrode fixing brackets, and an electrolytic cell. Both the anode and cathode are 304 stainless steel plates, and the electrode spacing is fixed at 10 cm. A pH meter is used to measure the pH during the electrocoagulation process.

[0032] 2.2 Electrocoagulation Treatment 1: This step aims to dynamically evaluate the initial floc formation rate under different conditions. Take an equal volume of algal solution (OD...) 680 The electrode (approximately 0.8) was placed in the electrocoagulation device and operated in constant current mode (1A). The electrode spacing (5cm) was kept consistent with the energizing time (the floc formation rate was calculated after 10 minutes of initial energizing) to ensure the comparability of the floc formation process under different treatment conditions. A rapid sample was taken exactly 10 minutes after energizing to determine the floc formation rate at this time (calculated according to the formula in Section 2.6), which served as an indicator for evaluating the initial flocculation reaction efficiency under different conditions.

[0033] 2.3 Electrocoagulation Treatment 2: Based on the above kinetic assessment, to obtain a large quantity of high-concentration microalgae for subsequent extraction operations, this step employs a complete harvest process. 50 mL of algal solution is added to a small electrocoagulation reactor, and the mixture is stirred periodically to ensure system homogeneity. Stainless steel electrodes are inserted and connected to a DC power supply. The initial pH (approximately 7.2–7.5) is recorded before the experiment begins. Power is then applied (1 A DC) and maintained in a constant current mode. After electrocoagulation begins, bubble generation, floc formation, and sedimentation occur. The reaction continues for 20–30 minutes until the pH approaches 8.8–9.0, at which point algal cell flocculation and sedimentation are essentially complete (the algal solution becomes clear and transparent except for the microalgae precipitate). The flocculated algal solution from the bottom of the electrolytic cell is collected and washed three times with pure water to obtain concentrated algal solution for subsequent solid-liquid separation (Section 2.5).

[0034] 2.4 pH Feedback Control (1) No pH control: only power is applied and pH changes over time are recorded naturally.

[0035] (2) Maintaining pH within the neutral range: The target pH range is 6.5-7.5. Adjust the pH by adding acid or alkali solution according to the data measured by the pH meter. When the pH exceeds 7.5, add dilute hydrochloric acid (0.1 mol / L); when the pH is below 6.5, add dilute sodium hydroxide (0.1 mol / L). The adjustment process should be run continuously as much as possible to maintain the pH of the system within the set range.

[0036] (3) Control the pH alkalinity range: The target pH range is 8.0-9.0. Alkali solution is added to adjust the pH based on the data measured by the pH meter. When the pH is below 7.5, dilute sodium hydroxide (0.1 mol / L) is added dropwise. When the pH is above 9.0, dilute hydrochloric acid (0.1 mol / L) is added dropwise. The adjustment process should be run as continuously as possible to maintain the pH of the system within the alkaline range.

[0037] 2.5 Solid-liquid separation process The concentrated algal solution after flocculation was then subjected to the following operations: (1) RT-centrifugation: The algal liquid after flocculation treatment is placed in a centrifuge and centrifuged at 25 ℃ with fixed parameters (8000 rpm, centrifugation time 15 min). The wet algal mud is collected and its weight is accurately recorded.

[0038] (2) Low temperature assisted centrifugation (4 ℃): After precooling the flocculated algal liquid at 4 ℃ for 20-30 min, place it in a centrifuge and separate the solid and liquid under the same parameters (8000 rpm, centrifugation time 15 min) at 4 ℃, and record the weight of the wet algal mud.

[0039] (3) Vacuum filtration: Pour the algal solution treated by electrocoagulation into the Buchner funnel and continue filtration until no obvious free water drips down. Record the weight of the resulting wet algal mud.

[0040] 2.6 Indicator Measurement and Calculation (1) Harvested biomass (g / L) Harvested biomass is defined as the final dry algal biomass (g / L) obtained per liter of the original algal solution. A sample of the moist algal sludge was dried at 60 ℃ to constant weight, and the dry mass was measured. Harvested biomass is an important indicator for evaluating microalgae harvesting efficiency. The pH change over time under different pH control conditions is shown below. Figure 1 As shown, the mean and standard deviation of harvested biomass under various conditions and at various time points are as follows: Figure 2 As shown in the figure. The results indicate that the highest harvest biomass was obtained under alkaline conditions with a pH of 8.0-9.0, suggesting that microalgae are more likely to flocculate and settle under weakly alkaline conditions, thereby improving harvest efficiency.

[0041] (2) Floc formation rate (%) in 10 min Floc formation rate is calculated as the ratio of the difference in algal cell concentration before and after 10 minutes of energization to the initial concentration: Floc formation rate (%) = (C0 - C 10 ) / C0 × 100%. Where C0 is the pre-flocculation algal cell concentration, C 10 The concentration of algal cells in the supernatant after 10 minutes of electrocoagulation was used to evaluate the floc formation rate and flocculation effect. Cell concentration was determined by dry weight method combined with UV absorbance. The floc formation rate reflects the aggregation rate and efficiency of algal cells during electrocoagulation; for example... Figure 3 As shown, the floc formation rate was highest under alkaline conditions, indicating that floc formation was faster at high pH. Under neutral and uncontrolled pH conditions, the floc formation rate was relatively low, sedimentation was slower, and harvesting efficiency decreased accordingly. This result suggests that in the electrocoagulation process under alkaline conditions, appropriately increasing the pH of the system helps to improve the biomass harvesting efficiency of *Micrococcus pluvialis*.

[0042] (3) Concentration factor Concentration factor = V0 / V1. Where V0 is the initial volume of the algal solution, and V1 is the volume of the wet algal sludge obtained after solid-liquid separation. The concentration factor reflects the degree of compression of the algal solution during the solid-liquid separation stage; a higher value indicates a more concentrated algal sludge. Figure 4 As shown, low-temperature assisted centrifugation can significantly increase the concentration factor, which is superior to room-temperature centrifugation and vacuum filtration. This result indicates that low-temperature treatment helps to promote the compaction and compression of algal sludge; its mechanism lies in the fact that low temperature can reduce the activity of algal cells and the viscosity of the system liquid, thereby making the solid-liquid separation process more thorough.

[0043] (4) Moisture content of mud (%) Weigh the wet algae mud, dry it to constant weight, and measure the dry mass. The mud moisture content is calculated as the proportion of water mass in the wet algae mud to the total mass of the wet mud: Mud Moisture Content (%) = (W wet - W dry ) / W wet × 100%. The mud moisture content represents the residual moisture in the wet algae mud; the lower the value, the better for subsequent drying. For example... Figure 5 As shown, low-temperature assisted centrifugation is the method with the lowest mud moisture content, which can effectively reduce the moisture content of algal mud.

[0044] 2.7 Algae Powder Preparation: The harvested algae mud was pre-frozen (-20℃), freeze-dried (-80℃), and then dried (60℃), and ground into uniform algae powder, which was then sealed and stored for later use. This algae powder, obtained based on the optimal harvesting scheme, will serve as the standardized raw material for the cell wall disruption pretreatment in Example 3 and the extraction of antibacterial active ingredients in Example 4.

[0045] In summary, alkaline environment conditioning can significantly improve the floc formation rate and biomass harvest rate of electrocoagulation. Low-temperature assisted centrifugation performs optimally in terms of concentration factor and moisture content control, yielding low-moisture algal sludge that facilitates subsequent drying and improves overall harvest efficiency. Therefore, electrocoagulation combined with low-temperature centrifugation under alkaline conditions is the best combination for microalgae harvesting. This approach balances harvest yield and solid-liquid separation efficiency, providing a technical reference for the preparation of core raw materials (algae powder) for the antibacterial extract of *Microcystis aeruginosa*.

[0046] Example 3: Screening of Preprocessing Methods Microchlorophyll cells possess a robust cell wall structure, making it difficult to effectively release valuable compounds (especially potential antibacterial active ingredients) using traditional solvent extraction methods. To overcome the limitations of the cell wall barrier on extraction efficiency, this invention screened five different pretreatment strategies, including: chemically assisted methods (acid pretreatment AP, alkali pretreatment BP), physically assisted methods (microwave pretreatment MP, ultrasonic pretreatment UP), and no pretreatment (NP).

[0047] (1) No Pretreatment (NP): Weigh 2 g of *Phyllostachys pulmonale* powder into a 200 mL beaker, add 50 mL of 75% ethanol, extract with a magnetic stirrer for 1 h, filter and repeat the above steps three times, collect and combine the liquid phases, evaporate to dryness in a water bath at 80 °C, weigh, and temporarily store in a centrifuge tube.

[0048] (2) Acid Pretreatment (AP): Weigh 2 g of *Phyllostachys pulmonale* powder into a 200 mL beaker, add 5 mL of 2 mol / L HCl and 45 mL of 75% ethanol, extract with a magnetic stirrer for 1 h, filter and repeat the above steps three times, collect and combine the liquid phases, evaporate to dryness in a water bath at 80 °C, weigh, and temporarily store in a centrifuge tube.

[0049] (3) Base Pretreatment (BP): Weigh 2 g of *Phyllostachys pulmonale* powder into a 200 mL beaker, add 5 mL of 2 mol / L NaOH and 45 mL of 75% ethanol, extract with a magnetic stirrer for 1 h, filter and repeat the above steps three times, collect and combine the liquid phases and adjust the pH to 7, evaporate to dryness in a water bath at 80 °C, redissolve the solids with anhydrous ethanol, filter and evaporate to dryness in a water bath at 80 °C, weigh, and temporarily store in a centrifuge tube.

[0050] (4) Microwave Pretreatment (MP): Weigh 2 g of *Phyllostachys pulmonale* powder into a 200 mL beaker, add 50 mL of 75% ethanol and stir to moisten. Place in a microwave oven and microwave for a total of 10 minutes at 700 W (1 min microwave treatment, 1 min interval, repeated 10 times). Extract with a magnetic stirrer for 1 h, filter and repeat the above steps three times, collect and combine the liquid phases, evaporate to dryness in a water bath at 80 °C, weigh, and temporarily store in a blue-capped centrifuge tube.

[0051] (5) Ultrasonic Pretreatment (UP): Weigh 2 g of *Phyllostachys pulmonae* powder into a 200 mL beaker, add 50 mL of 75% ethanol and mix well. Place the beaker into an ultrasonic cell disruptor, insert the ultrasonic probe of the ultrasonic instrument into the center of the suspension, run the ultrasonic pulse mode with 2 seconds on and 1 second off, power at 70%, and treat for 10 min. Then place the beaker in a magnetic stirrer to extract for 1 h, filter and repeat the above steps three times, collect and combine the liquid phases, evaporate to dryness in a water bath at 80 °C, weigh, and temporarily store in a blue-capped centrifuge tube.

[0052] Extract yields corresponding to different pretreatment methods for *Vibrio globosum* powder are as follows: Figure 6 As shown in the figure, ultrasonic pretreatment (UP) exhibited the highest extraction yield, significantly outperforming microwave pretreatment, chemical pretreatment, and conventional pretreatment without pretreatment in improving extraction efficiency. Therefore, considering the overall extraction efficiency, ultrasonic pretreatment was selected as the optimal pretreatment method. This optimal pretreatment method (ultrasonic pretreatment) will be directly applied to the final antibacterial substance extraction process in Example 4.

[0053] Example 4: Extraction of active extract from *Vibrio chlorella* 4.1 Preparation of Ethanol Extract from *Chlorella vulgaris* (1) Sample preparation and cell disruption: Weigh 10 g of *Vibrio chlorella* powder, add 160 mL of 75% ethanol solution, and mix thoroughly at room temperature. Then, sonicate the suspension (the maximum power of the sonic cell disruptor is 650 W, using a 2-second on / 1-second off pulse mode, power 70%, for 10 min) to disrupt the cell wall and release intracellular active components.

[0054] (2) Protease and DNase treatment: Filter the ultrasonically broken algal solution, retaining the initial ethanol extract (filtrate). Add 10 mL of pure water and 10 U of protease and DNase to the filtered algal powder (filter residue), and stir at room temperature for 30 min to degrade protein and nucleic acid impurities and reduce interference with low molecular weight compounds. After treatment, filter again, retain the filtered algal powder, and collect the aqueous phase for recycling in subsequent batches (enzyme pretreatment step for other batches of *Pheromonemus spp.* algal powder extraction. Reusing enzymes saves costs).

[0055] (3) Ethanol extraction: The enzyme-treated algal powder was extracted using the ethanol initial extract retained in step (2) (1 mL of ethanol initial extract corresponds to 1.4 g of algal powder); the extraction was repeated twice at 35°C, each time for about 1.5 hours, and stirred at 1200 rpm to ensure that the low molecular weight compounds were fully dissolved and decolorized.

[0056] (4) Filtration and preservation: Filter the extract to separate the algal residue from the liquid phase. Store the liquid phase in a brown bottle and wrap it with tin foil to prevent photodegradation.

[0057] (5) Evaporation and drying and product collection: The liquid phase was transferred to a 400 mL evaporating dish and evaporated in a water bath at 80 °C. The solid extract was collected and then freeze-dried to obtain a preliminarily concentrated low molecular weight compound extract, in which *Microchlorophyll* appeared as black crystals and dark green oil.

[0058] 4.2 Preparation of Ethanol-Alkali Co-extract from *Chlorella vulgaris* (1) Sample preparation and ultrasonic disruption: Weigh 10 g of *Pheromoneus globulus* powder and add 160 mL of a mixture, which is a 1:9 volume ratio of 2% NaOH and 75% ethanol, and mix thoroughly at room temperature. The resulting suspension is ultrasonically disrupted (the ultrasonic cell disruptor has a maximum power of 650 W, operates in a 2-second on / 1-second off pulse mode, at 70% power, for 10 min) to break down the cell wall and release intracellular substances.

[0059] (2) Protease and DNA enzyme treatment: Similar to the ethanol extraction method, the algal liquid after enzyme treatment is filtered, the filtered algal powder is retained, and the aqueous phase is collected for recycling (the enzyme pretreatment step for other batches of Microchlorophyll algae powder extraction work, recycling enzymes, saving costs), and the loss of extract is negligible.

[0060] (3) Ethanol-alkali co-extraction: The ethanol-alkali pre-extraction solution retained in step (2) was used to extract the enzyme-treated algal powder (1 mL of ethanol pre-extraction solution corresponds to 1.4 g of algal powder); the extraction was repeated twice at 35°C, each time for about 1.5 hours, with a stirring speed of 1200 rpm, to fully dissolve the low molecular weight compounds and decolorize them.

[0061] (4) Neutralization, filtration and preservation: Collect the liquid extract, neutralize it with 2% dilute hydrochloric acid, transfer it to an evaporating dish, evaporate and dry it in an 80°C water bath, dissolve the organic phase with anhydrous ethanol and separate NaCl by filtration, and store the resulting liquid in a brown bottle protected from light.

[0062] (5) Evaporation and product collection: The liquid phase was transferred to a 400 mL evaporating dish and evaporated in an 80 °C water bath. The black crystalline solid was collected and then freeze-dried to obtain a preliminary concentrated low molecular weight compound extract obtained by ethanol-alkali co-extraction. This extract has significant antibacterial activity against Candida albicans while retaining key active components such as fatty acids, flavonoids, and alkaloids.

[0063] Example 5: 5.1 Source of test strains: The test strains used in this experiment included Escherichia coli (No. 10907), Staphylococcus aureus (No. 10001), and Pseudomonas aeruginosa (No. 10204), all purchased from the China Industrial Microbial Culture Collection Center (CICC); Candida albicans (No. 10231) was purchased from the American Type Culture Collection (ATCC).

[0064] 5.2. Experimental Objective The evaluation of the antibacterial effect of the microalgae antibacterial active extract in this embodiment was based on the traditional understanding and efficient extraction of microalgae bioactive components. Broad-spectrum pathogenic bacteria (such as Escherichia coli and Staphylococcus aureus), typical pathogenic fungi (such as Candida albicans), and typical odor-producing bacteria (Pseudomonas aeruginosa) were used as test bacteria to determine their minimum inhibitory concentration (MIC). Colony counts (CFU) were counted using a gradient dilution method (1 to 400 times dilution). The antibacterial effects of the *P. chlorella* ethanol-alkali co-extract (hereinafter referred to as the ethanol-alkali co-extract) and the *P. chlorella* ethanol extract (hereinafter referred to as the ethanol extract) at various concentrations were compared and analyzed. The results showed a significant difference between the two extracts, verifying the advantages of the ethanol-alkali co-extraction method.

[0065] 5.3 Experimental Materials and Preparation This embodiment uses *Chlorella vulgaris* ethanol extract and *Chlorella vulgaris* ethanol-alkali co-extract as test samples to determine their antibacterial effects against specific test strains. Before the experiment, the test bacterial suspension was first diluted with phosphate-buffered saline (PBS) to prepare a concentration of 1×10⁻⁶. 4 CFU / mL up to 9×10 4 The test bacterial working solution is prepared at CFU / mL. Simultaneously, the test sample and control sample (such as solvent or blank solution) are diluted separately with standard hard water to the required test concentration.

[0066] 5.4 Antibacterial Contact and Neutralization Add 0.5 mL of the test bacterial working solution to sterile test tubes containing 5.0 mL of test sample dilution (each prepared with an initial concentration of 0.04 g / mL as either the *Pheretima asiatica* ethanol extract or the *Pheretima asiatica* ethanol-alkali co-extract, and serially diluted 10, 100, 200, 300, and 400 times to obtain different concentrations of test solution) and control sample dilution. Mix immediately and rapidly, and incubate at 20°C for 5 minutes. After the set incubation time, immediately transfer 0.5 mL of the mixture to a test tube containing 4.5 mL of sterile PBS, mix thoroughly, and terminate the continuous action of the test sample on the bacteria.

[0067] 5.5 Serial Dilution and Culture Counting Take 1 mL of the above dilution and inoculate it into two sterile Petri dishes. Then, pour 15 to 20 mL of nutrient agar medium cooled to 45°C into each dish (Luria-Bertani agar for bacterial tests, Sabouraud agar for fungal tests). Immediately after pouring, gently shake to ensure thorough mixing of the bacterial culture and the medium. After the agar has solidified, invert the Petri dishes and incubate them in a constant temperature incubator: incubate at 36±1°C for 48±2 hours for bacterial tests; incubate at 25±1°C for 72±3 hours for fungal tests. After incubation, count the colonies grown in the Petri dishes; if no colonies grow in the lower dilution dishes, record the colony count of the highest dilution dish where no colonies grow. The experiment is repeated three times in parallel, and the arithmetic mean of the data is calculated.

[0068] 5.6 Analysis of Antibacterial Effect Evaluation Results (1) Antibacterial activity against Escherichia coli like Figure 7 As shown, both extracts exhibited strong antibacterial activity at low dilutions (1x and 10x), with significantly lower colony counts than the control group (>400 CFU). The difference between the two gradually widened with increasing dilution: the ethanol extract still showed some inhibitory effect at a 100x dilution (average colony count 11 CFU), but its antibacterial ability decreased significantly at a 200x dilution, with the average colony count increasing to 130 CFU, and it essentially lost its antibacterial activity at a 400x dilution (>350 CFU). The ethanol-alkali co-extract, however, showed a sustained and highly effective inhibitory effect. Even at a dilution as high as 400x, with an average colony count of only 13 CFU, the antibacterial rate remained at an extremely high level, with a MIC of 0.4 mg / mL. This is attributed to the alkaline environment disrupting the integrity of the microalgal cell wall, promoting the release of more antibacterial components, which can still effectively disrupt the cell membrane structure of *E. coli* or inhibit its metabolism even at low concentrations.

[0069] (2) Antibacterial activity against Staphylococcus aureus like Figure 8As shown, both extracts exhibited significant concentration-dependent inhibition against Gram-positive bacteria: the ethanol extract had a narrow effective inhibition window, showing good efficacy up to 10-fold dilution (<3 CFU), but its antibacterial ability significantly weakened at 100-fold dilution, with the average colony count increasing to 58 CFU. The co-extract showed stronger antibacterial efficacy, maintaining an average colony count of 0 CFU at 100-fold dilution, achieving complete inhibition; antibacterial inactivation only occurred at 200-fold dilution, with a MIC of 0.4 mg / mL. Comparative data showed that the effective activity concentration range of the co-extract against Staphylococcus aureus was at least 10 times that of the ethanol extract (significant difference at the 100-fold dilution). This indicates that the ethanol-alkali co-extraction method significantly improves the bioavailability and bactericidal efficacy of the extract, superior to traditional single organic solvent extraction methods.

[0070] (3) Antibacterial activity against Candida albicans like Figure 9 As shown, the ethanol extract was essentially ineffective against Candida albicans. From 1-fold dilution (highest concentration) to 400-fold dilution, the colony count in the experimental group was >200, similar to the control group. The co-extract, however, exhibited extremely strong antifungal activity. In the 1-fold to 100-fold dilution range, the colony count was 0, completely inhibiting the growth of Candida albicans; only after 200-fold dilution did a small number of colonies appear (approximately 29.5 CFU), but this was still significantly lower than the control group, with a MIC of 0.2 mg / mL. This result reveals that the ethanol-alkali co-extract possesses unique broad-spectrum activity, particularly its antifungal ability lacking in the ethanol extract. Fungal cell walls are typically difficult to disrupt; alkaline treatment increases the dissolution rate of active substances from microalgae or protects their antibacterial active sites, giving the extract overall inhibitory ability against fungi. This is one of the most valuable findings obtained in this study through improved extraction processes.

[0071] (4) Antibacterial activity against odor-producing bacteria Pseudomonas aeruginosa Pseudomonas aeruginosa is a common odor-producing and opportunistic pathogen in daily life. Evaluation of its antibacterial effects shows that the extract can achieve deodorization functions in the daily chemical industry by inhibiting odor-producing bacteria. For example... Figure 10 As shown, the ethanol-alkali co-extract of *Pseudomonas aeruginosa* exhibits a significant bactericidal effect against this bacterium, with its effective antibacterial concentration range maintained up to 100-fold dilution (MIC approximately 0.4 mg / mL). Within its effective usage concentration range, the extract shows an overall inhibitory trend against the typical odor-producing bacterium *Pseudomonas aeruginosa*. Based on the control of odor-producing microorganisms such as *Pseudomonas aeruginosa*, the extract of this invention can effectively block the generation of everyday odors when combined with daily chemical products, providing a new natural odor-suppressing pathway for the development of deodorizing formulations.

[0072] In summary, the *Chlorella vulgaris* harvesting process and the ethanol-alkali co-extraction preparation process of this invention effectively improve the broad-spectrum antibacterial activity and purity of the extract, especially demonstrating outstanding antifungal properties. It can serve as a high-performance natural broad-spectrum antibacterial agent with significant industrialization value. The minimum inhibitory concentration (MIC) was quantitatively evaluated using the suspension method against broad-spectrum pathogens such as *Escherichia coli*, *Staphylococcus aureus*, *Candida albicans*, and odor-producing bacteria *Pseudomonas aeruginosa*. The results showed that the *Chlorella vulgaris* ethanol-alkali co-extract has a significant inhibitory effect on all of the above microorganisms, and its efficacy is significantly superior to that of the *Chlorella vulgaris* ethanol extract prepared by traditional extraction methods. This invention provides a new direction for developing novel natural antibacterial agents that combine enhanced antibacterial capabilities in daily chemical products with odor-eliminating and odor-inhibiting functions.

[0073] The embodiments of the present invention have been described in detail above, but the present invention is not limited to the described embodiments. For those skilled in the art, various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention, and these variations still fall within the protection scope of the present invention.

Claims

1. An extraction process for an active extract of *Vibrio chlorella*, characterized in that, Includes the following steps: S1. Harvesting of *Periplaneta micrantha*: The *Periplaneta micrantha* algal solution was electrocoagulated using an electrocoagulation device. During the electrocoagulation process, the algal solution was maintained in an alkaline environment with a pH of 8.0-9.

0. After sedimentation, the concentrated algal solution of the flocculated precipitate was taken, washed, and then separated into solid and liquid by low-temperature assisted centrifugation. The resulting wet algal mud was then pre-frozen, freeze-dried, and dried to obtain the final product. S2. Extraction of active extract of *Chlorella vulgaris*: Add ethanol-alkali mixed solution to *Chlorella vulgaris* obtained in S1, pre-treat with ultrasound, filter and collect the initial extract, treat the microalgae filter residue with protease and DNase, then use the collected initial extract to extract the enzyme-treated microalgae, collect the liquid extract, neutralize with hydrochloric acid and evaporate to dry, then dissolve the organic phase with ethanol, and the resulting liquid is evaporated and lyophilized to obtain the final product.

2. The extraction process of the active extract of *Pheretima aspergillum* according to claim 1, characterized in that, In S1, the temperature of the low-temperature assisted centrifugation is 0-4℃, the rotation speed is 8000 rpm, and the centrifugation time is 15 min.

3. The extraction process of the active extract of *Pheretima aspergillum* according to claim 1, characterized in that, In S1, the electrocoagulation time is 20-30 min.

4. The extraction process of the active extract of *Micrococcus pluvialis* according to claim 1, characterized in that, In S1, the electrocoagulation device includes a DC power supply, stainless steel electrode plates, electrode fixing brackets, and an electrolytic cell. Both the anode and cathode are stainless steel electrode plates, the electrode spacing is 7-15 cm, and the output current of the DC power supply is 1-3 A.

5. The extraction process of the active extract of *Micrococcus pluvialis* according to claim 1, characterized in that, In S2, the ethanol-alkali mixed solution is prepared by mixing 2% NaOH and 75% ethanol in a volume ratio of 1:7-11.

6. The extraction process of the active extract of *Micrococcus pluvialis* according to claim 1, characterized in that, In S2, the ultrasonic pretreatment is performed in a 2-3 second on / 1-2 second off pulse mode, with a power of 400-500 W and a time of 8-15 min.

7. The extraction process of the active extract of *Micrococcus pluvialis* according to claim 1, characterized in that, In S2, the enzyme-treated microalgae are extracted with the collected initial extract under stirring conditions. The extraction is repeated 2-4 times, each time for 1-3 hours.

8. The active extract of *Vibrio hygroscopicus* prepared by the extraction process according to any one of claims 1-7.

9. The use of the active extract of *Vibrio chlorella* as described in claim 8 in the preparation of products for antibacterial and / or Candida albicans treatment.

10. The application according to claim 9, characterized in that, The product includes antibacterial agents, deodorizers, or preservatives, and the bacteria include Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa.

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