A method for preserving euglena by using probiotics with low carbon source and narrow pH regulation

Abstract

The application discloses a kind of euglena biomass live preservation method and the product obtained.This method includes: the euglena slurry of harvesting is mixed with carbon source, buffer salt and prepared into fermentation medium, the carbon source concentration is 8-15 g / L;Then inoculate plant lactobacillus and lactobacillus acidophilus according to the volume ratio 2:1 compound probiotic seed liquid, fermentation is carried out at 30-33 DEG C for 36-48 hours, and in the fermentation process, by adding lye, pH is actively regulated and maintained at 5.8-6.2;After fermentation, euglena biomass is collected by centrifugation, and stored at 4 DEG C refrigeration.This application, through the synergistic effect of low carbon source concentration, narrow pH active regulation and specific double bacteria compound, prolongs the shelf life of euglena at 4 DEG C to more than 18 days, and the final live cell rate is maintained at more than 80%, while inducing aldehyde dehydrogenase to express efficiently, and the enzyme activity reaches 0.45 U / mg protein, realizing root odor removal, and the residual amount of hexanal is less than 2.0 μg / kg.This application effectively solves the technical problems of fresh euglena difficult to preserve for a long time and heavy odor, and has good industrial practicability.

Description

Technical Field

[0001] This invention belongs to the field of microalgae biological preservation technology, specifically relating to a method for preserving low concentrations of Euglena arvense in vivo by using a combination of Lactobacillus plantarum and Lactobacillus acidophilus fermentation under conditions of low carbon source concentration and narrow pH range active control. Background Technology

[0002] Euglena gracilis, also known as the slender euglena, is a single-celled eukaryotic microalga without a rigid cell wall, consisting only of a protein outer membrane. Rich in β-1,3-glucan, all essential amino acids, polyunsaturated fatty acids (DHA, EPA), vitamins, and minerals, Euglena is hailed as an "all-around nutritional microalga" and has broad application prospects in functional foods, health products, and pharmaceuticals.

[0003] However, fresh Euglena faces two major technical bottlenecks in post-harvest storage: First, it is extremely prone to spoilage after harvesting, and the shelf life of untreated low-concentration Euglena under 4°C refrigeration is usually only one week; second, Euglena itself has a strong algal odor, mainly from volatile carbonyl compounds such as hexanal, 2-octenal, and 1-octen-3-ol produced by the oxidation and degradation of polyunsaturated fatty acids, which seriously affects consumer acceptance.

[0004] Fermentation of microalgae using probiotics such as lactic acid bacteria has been reported. For example, Chinese patent CN120322157A discloses a "method for preserving and transforming algae by lactic acid fermentation," which uses lactic acid bacteria to ferment fresh algae and achieves preservation through pH control. However, when this method is directly applied to Euglena, the lack of a cell wall makes its protein outer membrane highly sensitive to metabolites such as organic acids and bacteriocins. Experiments show that when Euglena undergoes lactic acid fermentation according to the method in CN120322157A (pH naturally drops to 4.0), the viable cell rate drops to 22% after 24 hours of fermentation, and all cells die after 7 days of storage. The sensory score is only 3.0, exhibiting a strong rancid taste, completely failing to achieve the purpose of live preservation. This proves that conventional lactic acid bacteria fermentation preservation methods cannot be directly applied to the fragile Euglena without cell wall protection.

[0005] Another existing technology, CN105076899A, discloses a preservation method that uses lactic acid bacteria fermentation broth mixed with algal sludge and adds carbon sources such as glucose to promote fermentation. This technology also teaches how to achieve a preservation effect by adding exogenous carbon sources to promote rapid acid production by lactic acid bacteria. However, this method does not propose any differentiated carbon source control or cell protection strategies specifically for the cell wall-less microalgae.

[0006] Therefore, existing technologies lack a solution that can effectively balance the conflicting needs of probiotics in inhibiting bacteria and protecting Euglena cells. The real technical challenge lies in how to utilize probiotics to inhibit putrefactive bacteria while controlling their metabolic activity within a range that does not cause irreversible damage to the fragile Euglena cells. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings of existing technologies and provide a method for the live preservation of low-concentration Euglena using a combination of *Lactobacillus plantarum* and *Lactobacillus acidophilus* fermentation under conditions of low carbon source concentration and narrow-range active pH control. Through systematic experiments, this invention has discovered that the successful live preservation of Euglena does not depend on the optimization of a single parameter, but rather on the synergistic combination of three technical features: low carbon source concentration, narrow-range active pH control, and a specific combination of two bacteria. These three elements are interdependent and indispensable, forming an inseparable technical whole.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] A method for preserving Euglena through fermentation using a probiotic compound fermented with a low carbon source and narrow pH range includes the following steps:

[0010] (1) Preparation of Euglena sludge: Harvest freshly cultured Euglena, wash and concentrate to obtain fresh Euglena sludge with a water content of 85% to 95%;

[0011] (2) Preparation of fermentation substrate: Add carbon source and buffer salt to the Euglena sludge from step (1), mix evenly to obtain fermentation substrate; the carbon source is one of glucose, fructose or sucrose, with a final concentration of 8-15 g / L; the buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration of 0.05-0.15 mol / L, so that the initial pH of the fermentation substrate is maintained at 6.0-6.8;

[0012] (3) Preparation of probiotic seed liquid: Lactobacillus plantarum and Lactobacillus acidophilus were activated separately to obtain a viable count of 1×10⁻⁶. 8 ~1×10 9 CFU / mL seed culture; mix the two seed cultures at a volume ratio of 2:1 to obtain a compound probiotic seed culture;

[0013] (4) Temperature-controlled fermentation and active pH regulation: The compound probiotic seed liquid from step (3) is inoculated into the fermentation substrate from step (2) at a volume ratio of 3% to 8%, and fermented at 30 to 33°C for 36 to 48 hours; during the fermentation process, the pH of the system is controlled within the range of 5.8 to 6.2 by adding alkaline solution throughout.

[0014] (5) Post-processing and storage: After fermentation, the fermentation mixture is centrifuged or filtered at 4°C to collect Euglena biomass, which is then stored at 4°C.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] Through systematic experiments, this invention has found that the successful preservation of Euglena live organisms does not depend on the optimization of a single parameter, but rather on the synergistic combination of three technical features: low carbon source concentration, narrow-range active pH regulation, and specific dual-strain compound. These three features are interdependent and indispensable, forming an inseparable technical whole.

[0017] Specifically, the present invention has the following beneficial effects:

[0018] (1) Long-term, high-vitality preservation of fragile live Euglena: This invention, through the synergistic effect of low carbon source (8-15 g / L), narrow-range active pH regulation (5.8-6.2), and a specific dual-strain combination (Lactobacillus plantarum: Lactobacillus acidophilus = 2:1), significantly extends the shelf life of low-concentration Euglena to more than 18 days under refrigeration conditions of 4℃, and the viable cell rate at the endpoint is still maintained above 80% (Example 1). In contrast, abandoning any one or two key features, such as not performing pH regulation (Comparative Example 5), using a single strain (Comparative Examples 7 and 8), or having excessively high / low carbon source concentrations (Comparative Examples 4 and 6), cannot achieve the 15-day shelf life target, and the viable cell rate is significantly reduced. Jin Zhengjun's Q-value calculation confirms that the combined effect of the three factors shows a significant synergistic effect (Q > 1.15), and its shelf life is better than any group with missing or deviated single factors, effectively filling the technical gap that fresh Euglena cannot be preserved for a long time.

[0019] (2) Achieved fundamental and thorough enzymatic deodorization: The low carbon source concentration and weakly acidic environment (pH 5.8–6.2) specified in this invention can efficiently induce high expression of aldehyde dehydrogenase (ALDH), which is related to deodorization function, in probiotics. In Example 1, the ALDH enzyme activity reached 0.45 U / mg protein, significantly higher than the 0.18 U / mg protein under high carbon source conditions (40 g / L, Comparative Example 6). Under these optimal enzyme activity conditions, the residual amount of hexanal, a representative odorant, decreased from the initial 125 μg / kg to < 2.0 μg / kg, achieving fundamental elimination of Euglena odor from a biochemical perspective. Comparative Example 2 further demonstrates that the deodorization effect of this invention utilizes the unique physiological structure of Euglena, which lacks a cell wall and allows free permeability between enzymes and substrates. This is species-specific and cannot be easily extended to other cell-walled microalgae.

[0020] (3) Significantly improved process operability and robustness: By controlling the carbon source at a moderately low level (8-15 g / L), the acid production rate of probiotics was effectively suppressed. As shown in Example 1, only 3 pH adjustment operations are required every 48 hours to maintain the pH stably within the target range of 5.8-6.2. In contrast, when using a high carbon source (40 g / L, Comparative Example 6), acid production was too rapid, the adjustment frequency surged to 8-9 times / 48h, the process window was extremely narrow, and the operational difficulty increased sharply. This invention effectively avoids the problems of sudden pH drops, frequent adjustments, and difficulty in stabilization, making precise pH control strategies feasible and significantly improving the industrial scale-up potential and production stability of the technical solution.

[0021] (4) Economical and practical with low overall cost: The concentration of the carbon source (glucose, fructose or sucrose) used in this invention is only 1 / 3 to 1 / 2 (10 g / L to 30 g / L) of that used in conventional lactic acid bacteria fermentation, and the pH control adopts a conventional buffer system, resulting in mild operating conditions. While achieving a longer shelf life and better quality, it reduces raw material costs and process control difficulty, demonstrating outstanding economic efficiency and good industrialization potential.

[0022] (5) Clear criteria and boundaries for parameter selection are provided: Through systematic comparative experiments (Examples 4, 5, 6 and Comparative Examples 1-8), this invention clarifies the optimal range of each key parameter and the negative effects of deviations. For example, a carbon source concentration below 5 g / L leads to insufficient probiotic growth (Comparative Example 4), while a concentration above 20 g / L causes pH dysregulation, osmotic pressure damage, and inhibition of deodorizing enzyme activity (Comparative Example 6); abandoning pH regulation or having an excessively wide regulation range leads to irreversible acidic damage to Euglena cells (Comparative Examples 1, 5); using a single bacterial species cannot simultaneously achieve rapid initiation of antibacterial action and long-term maintenance of protection (Comparative Examples 7, 8). These detailed data provide solid experimental support for the scope of protection of the claims and enhance the indisputability of the technical solution. Attached Figure Description

[0023] Figure 1 A process flow diagram of one embodiment of the present invention.

[0024] Figure 2 Example 4: Comprehensive effect curve of carbon source concentration on preservation effect.

[0025] Figure 3 Example 5: Effect of different pH control ranges on the viability of Euglena cells.

[0026] Figure 4 A schematic diagram showing the relationship between shelf life and complementary functions of microbial strains under different compounding ratios.

[0027] Figure 5A comparison of the change in viable cell rate of Euglena under different treatment conditions over time.

[0028] Figure 6 A graph showing the relationship between aldehyde dehydrogenase activity and carbon source concentration. Detailed Implementation

[0029] The present invention will now be described in detail with reference to embodiments and comparative examples. The following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0030] Example 1

[0031] 1. Preparation of Euglena sphaerophyte: Culture medium of Euglena slenderis in the logarithmic growth phase was collected, filtered through a 300-mesh nylon filter, washed three times with deionized water, and concentrated by centrifugation (3000 rpm, 5 min) to obtain fresh Euglena sphaerophyte with a water content of approximately 90%. The Euglena sphaerophyte cell concentration in the sphaerophyte was determined to be approximately 5 × 10⁻⁶ cells / mL using the trypan blue staining method. 6 cells / g, initial cell viability was 96%.

[0032] 2. Preparation of fermentation substrate: Take 100 g of Euglena sludge, add 200 mL of sterile deionized water, and disperse evenly. Add glucose (food grade) to a final concentration of 10 g / L. Add a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system (0.1 mol / L, pH 6.5) and stir evenly. The initial pH was measured to be 6.4.

[0033] 3. Preparation of probiotic seed culture: *Lactobacillus plantarum* (from the China General Microbiological Culture Collection Center, accession number CICC 24809) and *Lactobacillus acidophilus* KDB-03 (from the China General Microbiological Culture Collection Center, accession number CICC 6081) were inoculated into MRS liquid medium and incubated at 37℃ for 24 h, yielding viable counts of 4.5 × 10⁻⁶ cells / mL. 8 CFU / mL and 5.0×10 8 The seed culture contained CFU / mL. The two seed cultures were mixed at a volume ratio of 2:1 to obtain a compound seed culture with a viable count of approximately 4.8 × 10⁻⁶ cells / mL. 8 CFU / mL.

[0034] 4. Temperature-controlled fermentation and active pH adjustment: The compound seed culture was inoculated into the fermentation substrate at a rate of 5% (v / v) and fermented at 32℃ and 100 rpm. pH was monitored every 6 hours. When the pH dropped to 5.8, it was slowly adjusted to 6.2 using 1 mol / L NaOH solution. Fermentation lasted for 48 hours, during which three pH adjustments were performed, with the pH strictly controlled between 5.8 and 6.2 throughout the process.

[0035] 5. Post-processing and storage: After fermentation, the fermentation mixture was centrifuged at 4000 rpm for 10 min at 4℃ to collect Euglena biomass, which was then transferred to a sterile sealed container and stored at 4℃.

[0036] Example 2 (Verification of Carbon Source Type)

[0037] The steps are the same as in Example 1, except that fructose and sucrose are used instead of glucose, respectively, with a concentration of 10 g / L.

[0038] Example 3 (Verification of Carbon Source Concentration)

[0039] The steps are the same as in Example 1, except that verification experiments were conducted at two carbon source concentrations of 8 g / L and 15 g / L, respectively.

[0040] Example 4 (Verification Experiment of Carbon Source Concentration Gradient)

[0041] This embodiment systematically verifies the effect of carbon source concentration on preservation effect, providing sufficient experimental support for the "8-15 g / L" range limitation in the claims.

[0042] The steps are the same as in Example 1, except that the experiments were conducted at ten carbon source concentration gradients of 3, 5, 8, 10, 12, 15, 18, 20, 25, and 35 g / L, with three replicates for each concentration.

[0043] Example 5 (pH adjustment range refinement verification experiment)

[0044] This embodiment systematically verifies the effect of different pH control target ranges on preservation effect, providing sufficient experimental support for the range limitation of "5.8 to 6.2" in the claims.

[0045] The steps are the same as in Example 1, except that the following pH control target ranges are set respectively: no control (let it run naturally), 3.5–4.0, 4.0–4.5, 4.5–5.0, 5.0–5.5, 5.5–6.0, 5.8–6.2, 6.2–6.8, and 6.8–7.5. Three replicates are made for each condition.

[0046] Example 6 (Comparison and Verification Experiment of Compound Ratio)

[0047] This embodiment systematically verifies the effect of different compound ratios of Lactobacillus plantarum and Lactobacillus acidophilus on the preservation effect, providing sufficient experimental support for the limitation of "volume ratio 2:1" in the claims.

[0048] The steps are the same as in Example 1, except that the following compounding ratios (Lactobacillus plantarum: Lactobacillus acidophilus, v / v) are used respectively: Lactobacillus plantarum only (1:0), 3:1, 2:1, 1:1, 1:2, 1:3, and Lactobacillus acidophilus only (0:1). The total inoculum amount remains the same (5% v / v).

[0049] Example 7 (Applicability verification with different initial Euglena concentrations)

[0050] This embodiment verifies the applicability of the method of the present invention under different initial Euglena concentrations.

[0051] The steps are the same as in Example 1, except that the following different initial concentrations of Euglena culture systems are used: 5×10³, 1×10³, etc. 4 5×10 4 1×10 5 5×10 5 1×10 6 5×10 6 cells / mL. Carbon source concentration is fine-tuned according to algal concentration: algal concentration ≤ 1 × 10⁻⁶ cells / mL. 4 At cells / mL, glucose was 8 g / L, and algal concentration was 1×10⁻⁶. 4 ~1×10 6 When the number of cells / mL is 10 g / L, the algal concentration is ≥1×10⁻⁶. 6 The glucose concentration was 12 g / L at cells / mL.

[0052] Example 8 (Verification of the effect of storage temperature on preservation effect)

[0053] The steps are the same as in Example 1, except that after fermentation, the Euglena biomass is stored at -20℃, 0℃, 4℃, 10℃, 15℃, and 25℃ respectively.

[0054] Example 9 (Equivalence verification of different carbon source types)

[0055] The steps are the same as in Example 1, except that glucose, fructose, sucrose, maltose, lactose, and galactose are used as carbon sources, with a concentration of 10 g / L for each.

[0056] Example 10 (Verification of the equivalence of buffer salt type and concentration)

[0057] The steps are the same as in Example 1, except that the following buffer systems are used respectively: disodium hydrogen phosphate-sodium dihydrogen phosphate (0.05, 0.10, 0.15 mol / L) and citric acid-disodium hydrogen phosphate (0.10 mol / L).

[0058] Example 11 (Orthogonal verification of fermentation temperature and time)

[0059] The steps are the same as in Example 1, except that an orthogonal design is used, and two factors are set: fermentation temperature (28℃, 30℃, 32℃, 35℃, 37℃) and fermentation time (24h, 36h, 48h, 60h, 72h).

[0060] Example 12 (Optimization and Validation of Inoculation Volume)

[0061] The steps are the same as in Example 1, except that the inoculation amount of the compound seed solution is 1%, 2%, 3%, 5%, 8%, 10%, and 15% (v / v), respectively.

[0062] Comparative Example 1 (Euglena treated according to method CN120322157A)

[0063] Following the method disclosed in CN120322157A, lactic acid bacteria were used to ferment Euglena, with sufficient carbon source (30 g / L glucose) added, and the pH was allowed to decrease naturally without active pH adjustment.

[0064] Comparative Example 2 (using microalgae with cell walls as the subject of the method of the present invention)

[0065] Chlorella was used instead of Euglena, and the rest of the operation was exactly the same as in Example 1.

[0066] Comparative Example 3 (only buffer salts and carbon sources were added, without inoculation with probiotics)

[0067] The steps are the same as in Example 1, except that no probiotics are inoculated.

[0068] Comparative Example 4 (inoculation with compound probiotics + active pH regulation, but without the addition of a carbon source)

[0069] The steps are the same as in Example 1, except that glucose is not added.

[0070] Comparative Example 5 (inoculated with compound probiotics + low carbon source (10 g / L), but without active pH regulation)

[0071] The steps are the same as in Example 1, except that no pH adjustment is performed.

[0072] Comparative Example 6 (inoculated with compound probiotics + active pH regulation, but using a high carbon source (40 g / L))

[0073] The steps are the same as in Example 1, except that the amount of glucose added is 40 g / L.

[0074] Comparative Example 7 (using only Lactobacillus plantarum + low carbon source (10 g / L) + pH adjustment).

[0075] The steps are the same as in Example 1, except that only Lactobacillus plantarum is used.

[0076] Comparative Example 8 (using only Lactobacillus acidophilus + low carbon source (10 g / L) + pH adjustment).

[0077] The steps are the same as in Example 1, except that only Lactobacillus acidophilus is used.

[0078] In this embodiment, all experiments were repeated three times, and the data are expressed as the mean ± standard deviation of the three replicates. The data were analyzed for significance using one-way ANOVA and Duncan's test in SPSS (P < 0.05).

[0079] (a) Performance testing methods:

[0080] The performance testing methods of the present invention will be described in detail below.

[0081] 1. Standards for determining shelf life

[0082] The shelf life is determined based on the following three indicators. If any one of the indicators fails to meet the requirements, the shelf life is considered to have expired:

[0083] (1) Microbiological indicators: Total colony count <10 5 CFU / mL. The plate count method was used. 1 mL of sample was serially diluted 10-fold with physiological saline, and the appropriate dilution was plated onto plate counting agar medium. After incubation at 36±1℃ for 48±2 hours, the CFU / mL was counted. Two replicates were performed for each dilution, and the average result was taken.

[0084] (2) Sensory rating: ≥6 points. See Article 3 of this section for the rating method.

[0085] (3) Viable cell rate: ≥70%. The determination method is described in Section 2 of this chapter.

[0086] Example of shelf life determination: On day N of storage, the sensory score of the sample first falls below 6 points, the viable cell rate first falls below 70%, or the total bacterial count first exceeds 10. 5 If the concentration of CFU / mL is CFU / mL, then the shelf life of the sample is recorded as N-1 days.

[0087] 2. Viable cell rate determination

[0088] The viable cell rate of Euglena was determined using the trypan blue staining exclusion method.

[0089] (1) Preparation of staining solution: Weigh 0.4 g trypan blue powder (analytical grade), dissolve it in 100 mL phosphate buffer (0.01 mol / L, pH 7.4) to prepare 0.4% (w / v) trypan blue staining solution, filter it through a 0.22 μm filter membrane for sterilization, store it at 4℃ protected from light, and bring it back to room temperature before use.

[0090] (2) Sample preparation: Take the Euglena sample to be tested, gently shake and resuspend, and put 100 μL of algal solution into a 1.5 mL centrifuge tube.

[0091] (3) Staining: Add 100 μL of trypan blue staining solution (algal solution to staining solution volume ratio 1:1) to the sample, gently mix by pipetting, and incubate at room temperature in the dark for 3 minutes. The staining time should not exceed 5 minutes to avoid damage to live cells due to prolonged exposure to the staining solution.

[0092] (4) Counting: Take 10 μL of the stained sample and inject it into the groove of the counting chamber of the hemocytometer. Cover with a coverslip to avoid air bubbles. Observe and count under an optical microscope (400× objective). Live cells repel trypan blue dye due to their intact cell membranes and appear colorless and transparent or pale green; dead cells or cells with damaged cell membranes lose their ability to repel dye and are stained blue.

[0093] (5) Counting rules: Count all cells in the four corner squares (each large square contains 16 small squares). For cells on the lines, follow the principle of "counting the left but not the right, counting the top but not the bottom". Each sample should contain no less than 200 cells. If the number of cells in a large square exceeds 50, the sample should be diluted appropriately and recounted.

[0094] (6) Calculate:

[0095]

[0096] Take the average of three independent counts for each measurement. If the coefficient of variation (CV) of the three parallel counts exceeds 15%, resampling and counting are required.

[0097] (7) Staining and counting should be completed within 30 minutes after sample collection to avoid affecting the accuracy of viable cell rate if the sample is left for too long. If probiotics and Euglena coexist in the sample, they should be distinguished and counted according to morphological differences (Euglena cells are significantly larger than probiotics and are spindle-shaped or elliptical, with chloroplasts and a light green color).

[0098] 3. Sensory evaluation

[0099] (1) Selection and training of evaluators: Twenty sensory evaluators (half male and half female, aged 20-40) who have undergone systematic training will be selected. The training content includes: identification of typical characteristics of Euglena (normal color, algal smell, fermented aroma, putrid smell, etc.), training on the use of scoring scales, and training on standard sample control. The training period shall be no less than 2 weeks, with 2 training sessions per week, each lasting 1 hour. After the training, a blind sample test will be conducted. Only evaluators whose scoring deviation of the standard sample is within ±1 point are eligible to participate in the formal evaluation.

[0100] (2) Sample preparation: Remove the sample from the 4℃ refrigerator and allow it to return to room temperature (25±2℃). Place approximately 10 g of sample in a colorless, transparent glass container of uniform size, and code the container with three random numbers. Prepare three independent replicates for each sample.

[0101] (3) Evaluation environment: Sensory evaluation is conducted in a dedicated sensory evaluation room, with the indoor temperature controlled at 25±2℃, relative humidity at 50%-60%, uniform and soft lighting, and no odor interference. Evaluators complete the evaluation in an independent evaluation cubicle.

[0102] (4) Evaluation items and scoring criteria:

[0103] 8-10 (Excellent) It has absolutely no fishy or algae-like smell, but rather a pleasant fermented or light floral aroma. Bright green or emerald green, with a uniform color. Uniform and smooth, without layering or sedimentation. 6-8 points (Good) It has a slight algae-like smell, but it's not unpleasant and is acceptable. The green is slightly dull, but the color is generally uniform. Mostly uniform, with slight layering, can be shaken to mix. 4-6 points (average) A distinct algae-like smell or a slightly sour smell can be detected. Yellowish-green or dark green, uneven in color Obvious layering or small amount of sediment <4 points (unacceptable) A strong fishy, ​​rancid, or other putrid odor. Yellowish-brown or grayish-green, with obvious discoloration. Severe stratification, clumping, or large amount of sedimentation

[0104] The overall sensory score is calculated as (odor score + color score + texture score) / 3. The result is the average ± standard deviation of the scores from 20 evaluators. If any single item score for a sample is below 4, the sample is deemed sensorily unacceptable regardless of the overall score.

[0105] (5) Evaluation process: Evaluators must refrain from eating, drinking, and smoking for one hour before the evaluation. The number of samples evaluated each time shall not exceed 6. Rinse your mouth with warm water between every two samples and allow at least 2 minutes between each sample to eliminate any residual effects. After the evaluation is completed, the team leader shall collect the scoring sheets and summarize the data.

[0106] 4. Hexanal content determination

[0107] The content of key odor substances such as hexanal in fermentation broth was determined by headspace solid-phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS).

[0108] (1) Sample pretreatment: Accurately measure 6.0 mL of fermentation broth sample and place it in a 20 mL headspace vial. Add 3.0 g of sodium chloride (analytical grade) to promote the release of volatile substances. Add 10 μL of 2,4,6-trimethylpyridine (TMP) internal standard solution (concentration of 1.0 μg / mL, solvent of methanol) as a quantitative internal standard. Seal the headspace vial with an aluminum cap with a polytetrafluoroethylene silicone septum.

[0109] (2) SPME extraction conditions: The sealed headspace vial was placed on a constant-temperature heated magnetic stirrer and equilibrated in a 40°C water bath for 20 minutes at a stirring speed of 250 rpm. Then, the aged SPME fiber head (DVB / CAR / PDMS, 50 / 30 μm, Supelco) was inserted into the headspace vial, and the fiber head was pushed out to expose the sample to the headspace gas phase. Headspace adsorption extraction was performed in a 40°C water bath for 20 minutes. After extraction, the fiber head was retracted, and the SPME handle was immediately removed from the headspace vial.

[0110] (3) GC-MS analysis conditions: Insert the extracted SPME fiber head into the GC injection port, desorb at 250 °C for 3 minutes, and inject without splitting.

[0111] Chromatographic conditions: DB-5MS quartz capillary column (30 m × 0.25 mm × 0.25 μm); Temperature program: initial temperature 40℃, hold for 3 minutes, increase to 120℃ at 5℃ / min, then increase to 230℃ at 10℃ / min, hold for 5 minutes; Carrier gas: high-purity helium (purity ≥ 99.99%), flow rate 1.0 mL / min, constant flow mode.

[0112] Mass spectrometry conditions: Electron impact ion source (EI), electron energy 70 eV, ion source temperature 230℃, quadrupole temperature 150℃, interface temperature 280℃. Full scan mode, mass scan range m / z 35-350, scan rate 4.45 scans / s.

[0113] (4) Qualitative analysis: The mass spectra of each detected chromatographic peak were compared with the mass spectrometry database of the National Institute of Standards and Technology (NIST) (NIST 17). The identification results with a matching degree of ≥80% were confirmed. The retention time of the chromatographic peak was compared with the retention time of the standard (hexanal standard, purity ≥98%, Sigma-Aldrich) under the same analytical conditions for confirmation.

[0114] (5) Quantitative analysis: Quantification was performed using the internal standard method. Using 2,4,6-trimethylpyridine as the internal standard, standard solutions of hexanal were prepared at different concentration gradients (0.5, 1.0, 5.0, 10.0, 50.0, 100.0, 200.0 μg / L). The solutions were measured under the same analytical conditions as the samples. A standard curve was established with the ratio of the hexanal peak area to the internal standard peak area as the ordinate and the hexanal concentration as the abscissa. The correlation coefficient R² of the standard curve should be no less than 0.995.

[0115] Formula for calculating the hexanal content in an unknown sample:

[0116]

[0117] In the formula: A_hexanal is the peak area of ​​hexanal in the sample; A_internal standard is the peak area of ​​the internal standard; C_internal standard is the concentration of the internal standard solution; V_internal standard is the volume of the internal standard solution added; m_sample is the mass of the sample (kg).

[0118] 5. Aldehyde dehydrogenase activity assay

[0119] (1) Preparation of crude enzyme solution: Take 50 mL of co-fermentation broth, centrifuge at 4℃ and 4000 rpm for 10 minutes, and collect the cell pellet. Wash the cells twice with pre-cooled phosphate buffer (0.05 mol / L, pH 7.5), centrifuging at 4℃ and 4000 rpm for 5 minutes each time. Resuspend the washed cells in 5 mL of pre-cooled extraction buffer (0.05 mol / L Tris-HCl, pH 8.0, containing 1 mmol / L EDTA, 1 mmol / L DTT and 10% glycerol). Place the cell suspension in an ice bath and process it using an ultrasonic cell disruptor: power 200W, sonication for 3 seconds, interval for 5 seconds, total processing time 15 minutes. The entire process is completed in an ice bath to prevent thermal denaturation of the enzyme protein. After ultrasonic disruption, centrifuge at 4℃ and 12000 rpm for 20 minutes, collect the supernatant, which is the crude enzyme solution, aliquot and store at -80℃ for later use.

[0120] (2) Protein concentration determination: The protein concentration in the crude enzyme solution was determined using the Bradford method. Bovine serum albumin (BSA) was used as the standard to prepare standard solutions with concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 mg / mL. 20 μL of the standard or crude enzyme solution was added to a 96-well plate, followed by 200 μL of Bradford working solution (Coomassie Brilliant Blue G-250). After mixing, the solution was reacted at room temperature for 5 minutes, and the absorbance at 595 nm was measured. A standard curve was plotted against the concentration (R²≥0.99), and the protein concentration of the crude enzyme solution was calculated based on the standard curve.

[0121] (3) Enzyme activity assay: Aldehyde dehydrogenase activity was determined using the substrate-coenzyme monitoring method. The total volume of the reaction system was 200 μL, containing: 0.1 mol / L Tris-HCl buffer (pH 8.0), 1 mmol / L NAD⁺ (oxidized coenzyme I), 2 mmol / L acetaldehyde (substrate), and an appropriate amount of crude enzyme solution (protein amount approximately 20-50 μg). The reaction was initiated with the addition of acetaldehyde, and the increase in absorbance was immediately monitored continuously at a wavelength of 340 nm (NADH characteristic absorption peak), with records taken every 10 seconds for 10 minutes, and the temperature was controlled at 30℃. A blank control without substrate and a negative control without enzyme solution were also set up.

[0122] (4) Enzyme activity calculation: Enzyme activity was calculated based on the slope of the initial linear phase of the absorbance change at 340 nm over time. One unit of enzyme activity (U) is defined as the amount of enzyme required to catalyze the production of 1 μmol of NADH per minute under the above reaction conditions. The molar extinction coefficient ε of NADH... 340 = 6.22 L / (mmol·cm).

[0123]

[0124] Where: ΔA 340 / min is the rate of change of absorbance at 340 nm per minute; V_total is the total reaction volume (mL); ε is the NADH molar extinction coefficient; L is the optical path length (cm); V_enzyme is the volume of enzyme solution added (mL); C_protein is the protein concentration of the enzyme solution (mg / mL).

[0125] 6. Synergistic Effect Analysis

[0126] To quantitatively evaluate whether there is a synergistic effect among three technical characteristics—low carbon source concentration (factor A), active pH regulation (factor B), and specific two-drug combination (factor C)—the Jinzhengjun Q-value method was used. The Jinzhengjun Q-value method was initially used in pharmacology to quantitatively evaluate whether there are synergistic, additive, or antagonistic effects when two or more drugs are used in combination. Later, it was widely applied to determine the synergistic effect of multi-factor complexes.

[0127] If the effects of multiple factors on a certain effect index are independent (i.e., there is no synergy or antagonism), then their combined effect value (E_measured) should be equal to the expected combined effect value (E_expected) calculated by probability theory from the individual effect values ​​of each factor. When the measured combined effect value is significantly greater than the expected combined effect value, it suggests the existence of synergy; when the measured value is significantly less than the expected value, it suggests the existence of antagonism.

[0128] Calculation steps:

[0129] (1) Normalization of effect value: Using the shelf life of the untreated blank control group (i.e., the untreated blank control group, i.e., without carbon source, without probiotic inoculation, without fermentation, and directly refrigerated) as the baseline, the shelf life (days) under each factor combination condition was normalized to the effect value E:

[0130]

[0131] In the formula: T_x is the shelf life (days) of a certain treatment group; T_baseline is the shelf life (days) of the blank control group.

[0132] (2) Calculation of the expected joint effect value: According to the addition rule of probability theory, when the effects of factors A, B, and C are independent of each other, their joint expected effect value is:

[0133]

[0134] In the formula: E_A is the normalized effect value of factor A (low carbon source + compound, but no pH adjustment) only; E_B is the normalized effect value of factor B (narrow pH range + compound, but no carbon source) only; E_C is the normalized effect value of factor C (low carbon source + narrow pH range, but no compound).

[0135] The single-factor effect values ​​E_A, E_B, and E_C were obtained through experimental determination. Except for the missing factor, the experimental conditions were completely consistent with those in Example 1.

[0136] (3) Calculation of the measured combined effect value: Example 1 is a complete scheme in which all three factors A, B and C exist. Its normalized effect value E_measured is E_Example 1. The calculation method is the same as step (1).

[0137] (4) Calculation of Jin Zhengjun's Q value:

[0138]

[0139] (1) Result determination:

[0140] If Q > 1.15: There is a significant synergistic effect among factors A, B, and C, meaning that the combined effect of the three is higher than the expected value of their individual effects, suggesting a positive interaction effect among the three.

[0141] If 0.85 ≤ Q ≤ 1.15: Factors A, B, and C have an additive effect, meaning the combined effect equals the sum of the independent effects of each factor, and there is no interaction effect.

[0142] If Q < 0.85: There is an antagonistic effect among factors A, B, and C, that is, the combined effect is lower than the mathematical expectation of their individual effects, indicating the existence of a negative interaction effect.

[0143] The individual effect values ​​E_A, E_B, and E_C for each factor are from the corresponding response ratio experiment.

[0144] E_A represents the data from Comparative Example 5 (low carbon source + compound formulation, but without pH adjustment); E_B represents the data from Comparative Example 4 (narrow pH range + compound formulation, but without carbon source); E_C is the average of Comparative Examples 7 and 8 (low carbon source + narrow pH range, single bacterial species only); E_Measured represents the data from Example 1. For specific experimental conditions and results, please refer to the corresponding sections of the Examples and Comparative Examples.

[0145] Calculation example (data is hypothetical and used only to illustrate the method):

[0146] Assume the shelf life of the blank baseline is 3 days;

[0147] If the shelf life is 10 days when only factor A is considered, then E_A = (10-3) / 3 ≈ 2.333;

[0148] If the shelf life is 8 days when only factor B is considered, then E_B = (8-3) / 3 ≈ 1.667;

[0149] When only factor C is considered, the shelf life is 12.5 days (taking the average of comparative examples B5 and B6), then E_C = (12.5-3) / 3 ≈3.167;

[0150] Calculate E_expected = 2.333 + 1.667 + 3.167 - (2.333×1.667) - (1.667×3.167) - (2.333×3.167) + (2.333×1.667×3.167);

[0151] Calculate E_expected ≈ 4.500;

[0152] The actual shelf life of Example 1 was measured to be 18 days, E_actual = (18-3) / 3 = 5.000;

[0153] Q = 5.000 / 4.500 ≈ 1.111.

[0154] If the calculation result shows Q > 1.15, it can be determined as a significant synergistic effect.

[0155] (II) Results of efficacy verification and mechanism analysis:

[0156] The present invention will now be described in detail with reference to embodiments and comparative examples. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0157] Comparison of the overall effects of Example 1 and all other examples:

[0158] Example 1 (Best Solution)

[0159] 1. Preparation of Euglena sphaerophyte: Culture medium of Euglena slenderis in the logarithmic growth phase was collected, filtered through a 300-mesh nylon filter, washed three times with deionized water, and concentrated by centrifugation (3000 rpm, 5 min) to obtain fresh Euglena sphaerophyte with a water content of approximately 90%. The Euglena sphaerophyte cell concentration in the sphaerophyte was determined to be approximately 5 × 10⁻⁶ cells / mL using the trypan blue staining method. 6 cells / g, initial cell viability was 96%.

[0160] 2. Preparation of fermentation substrate: Take 100 g of Euglena sludge, add 200 mL of sterile deionized water, and disperse evenly. Add glucose (food grade) to a final concentration of 10 g / L. Add a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system (0.1 mol / L, pH 6.5) and stir evenly. The initial pH was measured to be 6.4.

[0161] 3. Preparation of probiotic seed culture: *Lactobacillus plantarum* (from China General Microbiological Culture Collection Center, accession number CICC 24809) and *Lactobacillus acidophilus* (from China General Microbiological Culture Collection Center, accession number CICC 6081) were inoculated into MRS liquid medium and incubated at 37℃ for 24 h, yielding viable counts of 4.5 × 10⁻⁶ cells / mL. 8 CFU / mL and 5.0×10 8 The seed culture contained CFU / mL. The two seed cultures were mixed at a volume ratio of 2:1 to obtain a compound seed culture with a viable count of approximately 4.8 × 10⁻⁶ cells / mL. 8 CFU / mL.

[0162] 4. Temperature-controlled fermentation and active pH adjustment: The compound seed culture was inoculated into the fermentation substrate at a rate of 5% (v / v) and fermented at 32℃ and 100 rpm. pH was monitored every 6 hours. When the pH dropped to 5.8, it was slowly adjusted to 6.2 using 1 mol / L NaOH solution. Fermentation lasted for 48 hours, during which three pH adjustments were performed, with the pH strictly controlled between 5.8 and 6.2 throughout the process.

[0163] 5. Post-processing and storage: After fermentation, the fermentation mixture was centrifuged at 4000 rpm for 10 min at 4℃ to collect Euglena biomass, which was then transferred to a sterile sealed container and stored at 4℃.

[0164] Example 1 Effect Test Results:

[0165] Shelf life is 18 days. The endpoint (day 18) showed a cell viability of 81%, an endpoint sensory score of 8.5, a hexanal content of < 2.0 μg / kg, a DPPH free radical scavenging rate that increased by 52% compared to before treatment, and an ALDH enzyme activity of 0.45 U / mg protein.

[0166] Example 2 (Verification of Carbon Source Type)

[0167] The steps are the same as in Example 1, except that fructose and sucrose are used instead of glucose, respectively, with a concentration of 10 g / L.

[0168] Table 1. Comparison of the effects of different carbon source types in Example 2

[0169] glucose 18 81 8.5 < 2.0 <![CDATA[3.0×10 8 ]]> fructose 17 79 8.2 < 2.5 <![CDATA[2.5×10 8 ]]> sucrose 16 77 8.0 < 3.0 <![CDATA[2.2×10 8 ]]>

[0170] Results analysis:

[0171] Glucose, fructose, and sucrose all achieved excellent preservation effects (shelf life > 15 days, viable cell rate > 75%), indicating that the range of carbon source types defined in the claims has good applicability. Among them, glucose showed the best effect, which may be related to the fact that *Lactobacillus plantarum* and *Lactobacillus acidophilus* have the highest efficiency in transporting and utilizing glucose.

[0172] Example 3 (Verification of Carbon Source Concentration)

[0173] The steps are the same as in Example 1, except that verification experiments were conducted at two carbon source concentrations of 8 g / L and 15 g / L, respectively.

[0174] Table 2. Validation effect of carbon source concentration in Example 3

[0175] 8 16 83 8.3 2 10 18 81 8.5 3 15 17 78 8.2 4

[0176] Results analysis:

[0177] Both groups achieved the technical goals of a shelf life of >15 days and a viable cell rate of >75%, further validating the effectiveness and operability of the carbon source concentration range of 8–15 g / L.

[0178] The 8 g / L group had the highest cell viability (83%), indicating that, under the premise of ensuring the basic metabolic needs of probiotics, the lower the carbon source concentration, the smaller the metabolic load on Euglena cells. However, the shelf life was slightly shorter than that of the 10 g / L group, suggesting that the long-term antibacterial ability of probiotics is slightly insufficient when the carbon source concentration is too low.

[0179] Example 4 (Verification Experiment of Carbon Source Concentration Gradient)

[0180] This embodiment systematically verifies the effect of carbon source concentration on preservation effect, providing sufficient experimental support for the "8-15 g / L" range limitation in the claims.

[0181] The steps are the same as in Example 1, except that the experiments were conducted at ten carbon source concentration gradients of 3, 5, 8, 10, 12, 15, 18, 20, 25, and 35 g / L, with three replicates for each concentration.

[0182] Table 3. Results of the carbon source concentration gradient experiment in Example 4

[0183] 3 6 80 0 5.0 68 Insufficient probiotic growth and poor antibacterial effect 5 9 79 1 6.0 32 The antibacterial effect was barely achieved, failing to reach the 15-day target. 8 16 83 2 8.3 <3.0 Excellent effect 10 18 81 3 8.5 <2.0 Optimal solution 12 18 80 3 8.4 <2.0 Excellent effect 15 17 78 4 8.2 <2.5 Excellent effect 18 14 70 5 7.2 <3.0 Increased difficulty in regulation, decreased viable cell rate 20 13 66 7 6.8 <3.0 Frequent adjustments and operational difficulties 25 12 60 10 6.2 8 Excessive regulation leads to significant infiltration stress. 35 10 52 15 5.5 12 The system went out of control, and the effect deteriorated significantly.

[0184] Results analysis:

[0185] (1) In the range of low carbon source concentration (3-5 g / L): the growth of probiotics is limited, and the number of viable bacteria after 48 hours is only 6.5×10. 6 CFU / mL (3 g / L) and 1.8×10 7CFU / mL (5 g / L), significantly lower than the 3.0 × 10⁻⁶ CFU / mL in Example 1. 8 The concentration was CFU / mL. The antibacterial effect was insufficient, and the shelf life was only 6–9 days, far from meeting practical preservation needs. This result verifies the scientific basis for setting the lower limit of carbon source concentration to 8 g / L in the claims.

[0186] (2) Optimal range (8–15 g / L): Within this range, all concentration groups achieved the technical goals of a shelf life of over 15 days and a viable cell rate of over 75%. As the carbon source concentration increased, the shelf life first increased and then decreased, reaching a peak of 18 days at 10 g / L; the viable cell rate continued to decrease, from 83% at 8 g / L to 78% at 15 g / L. The frequency of pH regulation increased with the increase of carbon source concentration, indicating that the metabolic activity of probiotics was enhanced. This range is precisely in the balance zone between "moderate growth of probiotics" and "controllable metabolic rate".

[0187] (3) In the range of excessively high carbon source concentration (18–35 g / L): the acid production rate increases, and the frequency of pH regulation surges from 5 times / 48h (18 g / L) to 15 times / 48h (35 g / L), leading to a tendency for the system to become uncontrollable. Cell viability continues to decline: when the carbon source concentration is ≥20 g / L, the cell viability drops below 66%; at 35 g / L, the cell viability is only 52%. High osmotic pressure causes additional stress to Euglena, which lacks a cell wall. Notably, the increase in carbon source concentration did not correspondingly improve shelf life or deodorization effect; instead, it caused the residual hexanal to rise from <2.0 μg / kg to 12 μg / kg. This result verifies the necessity of setting the upper limit of carbon source concentration to 15 g / L in the claims.

[0188] Example 5 (pH adjustment range refinement verification experiment)

[0189] This embodiment systematically verifies the effect of different pH control target ranges on preservation effect, providing sufficient experimental support for the range limitation of "5.8 to 6.2" in the claims.

[0190] The steps are the same as in Example 1, except that the following pH control target ranges are set respectively: ① No control (let it be natural); ② 3.5~4.0; ③ 4.0~4.5; ④ 4.5~5.0; ⑤ 5.0~5.5; ⑥ 5.5~6.0; ⑦ 5.8~6.2; ⑧ 6.2~6.8; ⑨ 6.8~7.5.

[0191] Table 4. Results of the pH control range refinement verification experiment in Example 5

[0192] No adjustment (down to 4.8) 10 65 5.8 18 The viable cell rate decreased significantly. 3.5～4.0 8 22 4.2 35 Severe acid damage, massive cell death 4.0～4.5 10 38 5.0 28 Excessive acidity leads to significant cell damage. 4.5～5.0 12 55 6.0 20 Below the tolerance limit of Euglena 5.0～5.5 14 68 6.5 12 The viable cell rate is less than 70%. 5.5～6.0 16 76 8.0 <3.0 Close to target but with slightly low live cell rate 5.8～6.2 18 81 8.5 <2.0 Optimal solution 6.2～6.8 14 82 7.0 8 Insufficient antibacterial properties 6.8～7.5 8 85 5.0 42 Insufficient antibacterial properties

[0193] Results analysis:

[0194] (1) Low pH range (<5.0): When pH≤4.5, Euglena suffers severe irreversible acidic damage, and the viable cell rate drops to below 38% (only 22% in the 3.5-4.0 group), with a shelf life of only 8-10 days. The sensory score also decreases (4.2-5.0 points), and a rancid odor appears.

[0195] (2) pH transition range (5.0-5.5): The viable cell rate recovers somewhat (55%-68%), but is still below the target lower limit of 70%. pH 5.0 is the key dividing line for Euglena tolerance; below this value, the viability drops sharply.

[0196] (3) Low pH range (5.5-6.0): Shelf life 16 days, endpoint viable cell rate 76%, which is close to but not as good as the optimal effect of Example 1. This indicates that there is still some acidic stress near the lower limit of pH, and there is room for further optimization of viable cell rate.

[0197] (4) Optimal range (5.8-6.2): ​​All indicators reached their optimal levels. The viable cell rate of Euglena was as high as 81%, and the ALDH enzyme activity was the highest (0.45 U / mg protein), making it the best pH window for achieving root deodorization.

[0198] (5) High pH range (>6.2): ​​Although the cell viability of Euglena remained good (82%~85%), the antibacterial effect was seriously insufficient. The shelf life of the 6.2~6.8 group and the 6.8~7.5 group was shortened to 14 days and 8 days respectively, indicating that when pH>6.5, spoilage bacteria multiplied in large numbers, and the system lost its preservation significance.

[0199] This result verifies the dual necessity of setting the upper limit of pH to 6.2 and the lower limit to 5.8 in the claims.

[0200] Example 6 (Comparison and Verification Experiment of Compound Ratio)

[0201] This embodiment systematically verifies the effect of different compound ratios of Lactobacillus plantarum and Lactobacillus acidophilus on the preservation effect, providing sufficient experimental support for the limitation of "volume ratio 2:1" in the claims.

[0202] The steps are the same as in Example 1, except that the following compounding ratios (Lactobacillus plantarum:Lactobacillus acidophilus, v / v) are used respectively: ①Lactobacillus plantarum only (1:0); ②3:1; ③2:1; ④1:1; ⑤1:2; ⑥1:3; ⑦Lactobacillus acidophilus only (0:1). The total inoculum amount is kept consistent (5% v / v), with three replicates per group.

[0203] Table 5. Results of the comparative verification experiment on the compounding ratio in Example 6

[0204] P only (1:0) 13 74 7.0 3:1 16 78 8.0 2:1 18 81 8.5 1:1 15 77 7.5 1:2 14 75 7.2 1:3 13 72 6.8 Only A (0:1) 12 71 6.5

[0205] Results analysis:

[0206] Lactobacillus acidophilus is the main force in rapidly producing acid and quickly establishing the initial pH antibacterial barrier during the early stage of fermentation. A 2:1 ratio is the optimal balance point: only when Lactobacillus plantarum and Lactobacillus acidophilus are combined in a 2:1 ratio does the optimal metabolic division of labor between rapid early-stage antibacterial activity and long-term protection in the later stages is achieved. The product has a shelf life of 18 days, a viable cell rate of 81%, and a sensory score of 8.5, all significantly better than all other formulation ratios.

[0207] Example 7 (Applicability verification with different initial Euglena concentrations)

[0208] This embodiment verifies the applicability of the method of the present invention under different initial Euglena concentrations, confirming the universality of the technical solution protected by the claims in the range of low-concentration Euglena.

[0209] The steps are the same as in Example 1, except that the following different initial concentrations of Euglena culture systems are used: 5×10³, 1×10³, etc. 4 5×10 4 1×10 5 5×10 5 1×10 6 5×10 6 cells / mL. Carbon source concentration is fine-tuned according to algal concentration: algal concentration ≤ 1 × 10⁻⁶ cells / mL. 4 At cells / mL, glucose was 8 g / L, and algal concentration was 1×10⁻⁶. 4 ~1×10 6 When the number of cells / mL is 10 g / L, the algal concentration is ≥1×10⁻⁶. 6 The glucose concentration was 12 g / L at cells / mL.

[0210] Table 6. Results of the experiment on the applicability of different initial Euglena concentrations in Example 7

[0211] 5×10³ 8 15 80 8.0 <3.0 <![CDATA[1×10 4 ]]> 8 16 82 8.2 <2.5 <![CDATA[5×10 4 ]]> 10 17 81 8.3 <2.5 <![CDATA[1×10 5 ]]> 10 18 81 8.5 <2.0 <![CDATA[5×10 5 ]]> 10 18 80 8.4 <2.0 <![CDATA[1×10 6 ]]> 12 17 80 8.2 <2.5 <![CDATA[5×10 6 ]]> 12 16 79 8.0 <3.0

[0212] Results analysis:

[0213] In 1×10 4 ~1×10 6 Within the low concentration range of Euglena cells / mL, the method of this invention can achieve the technical goals of a shelf life ≥15 days and a viable cell rate ≥75%, verifying the universality and robustness of the method within this concentration range. However, when the algal concentration is too low (5×10³ cells / mL) or too high (5×10³ cells / mL), the method will not achieve the desired results. 6 When the concentration of carbon source was adjusted to (cells / mL), the preservation effect could still be maintained by fine-tuning the carbon source concentration (within the range of 8–15 g / L), but the shelf life and viable cell rate decreased slightly, suggesting that the microecological balance of probiotics-Euglena was slightly affected under extremely low or extremely high algae concentrations.

[0214] Therefore, the method of the present invention is suitable for Euglena cell concentrations of 1×10⁻⁶. 4 ~1×10 6 Excellent technical results can be achieved within the range of cells / mL.

[0215] Example 8 (Verification of the effect of storage temperature on preservation effect)

[0216] This embodiment verifies the stability of the preservation effect of the present invention under different storage temperatures, and confirms that 4°C refrigeration is the preferred storage temperature as a scientific basis.

[0217] The steps are the same as in Example 1, except that after fermentation, the Euglena biomass is stored at -20℃, 0℃, 4℃, 10℃, 15℃, and 25℃ respectively. Each group has three replicates.

[0218] Table 7. Experimental Results on the Effect of Storage Temperature in Example 8

[0219] -20 >60 25 6.5 Freeze-thaw cycles cause cell rupture and result in a low cell viability rate. 0 25 84 8.0 Excellent performance, but requires continuous cooling. 4 18 81 8.5 Balancing effectiveness and cost-effectiveness, the optimal approach is... 10 12 76 7.5 The effect began to decline 15 8 68 6.8 The shelf life is significantly shortened. 25 4 45 5.0 Decomposition accelerates at room temperature

[0220] Results analysis:

[0221] 4℃ refrigeration is the optimal storage temperature balancing preservation effect and energy consumption cost. At 0℃, the shelf life is extended to 25 days, but the energy consumption increases due to the need for continuous refrigeration; although freezing at -20℃ can theoretically preserve for a long time, the freeze-thaw process causes a large number of Euglena cells to rupture due to ice crystal formation, resulting in a viable cell rate of only 25%, making it completely unsuitable for applications where the purpose is to preserve live cells. At temperatures of 10℃ and above, the shelf life is significantly shortened, and above 15℃, preservation is no longer meaningful.

[0222] Example 9 (Equivalence verification of different carbon source types)

[0223] This embodiment verifies the equivalence of the carbon source types (glucose, fructose, sucrose) described in the claims, and compares them with other common sugars.

[0224] The steps are the same as in Example 1, except that glucose, fructose, sucrose, maltose, lactose, and galactose are used as carbon sources, each at a concentration of 10 g / L. Three replicates are used for each group.

[0225] Table 8. Comparison of effects of different carbon source types in Example 9

[0226] glucose 18 81 <![CDATA[3.0×10 8 ]]> <2.0 Optimal, with the highest utilization efficiency fructose 17 79 <![CDATA[2.5×10 8 ]]> <2.5 Excellent, with effects similar to glucose. sucrose 16 77 <![CDATA[2.2×10 8 ]]> <3.0 Excellent, utilized after being broken down by sucrase. maltose 14 75 <![CDATA[1.8×10 8 ]]> 5 It is usable, but the effect is slightly reduced. lactose 10 74 <![CDATA[8.5×10 7 ]]> 18 Lactobacillus plantarum has low utilization efficiency. Galactose 9 73 <![CDATA[6.2×10 7 ]]> 22 Lactobacillus acidophilus has low utilization efficiency.

[0227] Results analysis:

[0228] Glucose, fructose, and sucrose can all achieve excellent preservation effects (shelf life > 15 days, live cell rate > 75%).

[0229] Glucose showed the best effect, likely due to its highest transport and phosphorylation efficiency within probiotics. While maltose could be utilized, the viable cell count after 48 hours was only 60% of that of the glucose group, indicating a slower utilization rate. Lactose and galactose showed significantly reduced utilization efficiency and noticeably deteriorated preservation effects due to the low activity of corresponding hydrolytic enzymes or transport proteins in *Lactobacillus plantarum* and *Lactobacillus acidophilus*.

[0230] This result validates the scientific basis for limiting the carbon source in the claim to "one of glucose, fructose or sucrose".

[0231] Example 10 (Verification of the equivalence of buffer salt type and concentration)

[0232] This embodiment verifies the applicability of the type and concentration of buffer salt described in the claims.

[0233] The steps are the same as in Example 1, except that the following buffer systems are used respectively: ① disodium hydrogen phosphate-sodium dihydrogen phosphate, 0.05 mol / L; ② disodium hydrogen phosphate-sodium dihydrogen phosphate, 0.10 mol / L; ③ disodium hydrogen phosphate-sodium dihydrogen phosphate, 0.15 mol / L; ④ citric acid-disodium hydrogen phosphate, 0.10 mol / L.

[0234] Table 9. Comparison of the effects of buffer salt type and concentration in Example 10

[0235] Phosphate buffer 0.05 16 80 5 8.2 Phosphate buffer 0.10 18 81 3 8.5 Phosphate buffer 0.15 17 82 2 8.3 Citric acid-phosphate 0.10 17 80 3 8.3

[0236] Results analysis:

[0237] The disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system effectively buffered pH within a concentration range of 0.05–0.15 mol / L, with a shelf life exceeding 15 days. At 0.05 mol / L, the buffering capacity was weaker, requiring pH adjustment frequency to increase to 5 times / 48h; at 0.15 mol / L, the frequency decreased to 2 times / 48h, but higher salt concentrations had a slight negative impact on Euglena cell viability (no significant difference between the 82% and 81% viability rates in the 0.10 mol / L group).

[0238] A concentration of 0.10 mol / L achieved an optimal balance between buffering efficiency and salt concentration. The citrate-phosphate buffer system was also feasible and performed similarly to the phosphate system.

[0239] Example 11 (Orthogonal verification of fermentation temperature and time)

[0240] This embodiment uses orthogonal experiments to verify the optimal combination of fermentation temperature and fermentation time.

[0241] The steps are the same as in Example 1, except that an orthogonal design is used, setting two factors: fermentation temperature (28℃, 30℃, 32℃, 35℃, 37℃) and fermentation time (24h, 36h, 48h, 60h, 72h), for a total of 25 combinations. Each group has three replicates.

[0242] Table 10. Results of the orthogonal experiment of fermentation temperature and time in Example 11 (shelf life / day)

[0243] 28℃ 9 12 14 15 14 30℃ 12 15 17 16 15 32℃ 14 17 18 17 16 35℃ 12 15 16 15 14 37℃ 10 13 14 13 12

[0244] Table 11. Viable cell percentage (%, storage endpoint) for different combinations

[0245] 28℃ 87 85 84 82 80 30℃ 85 83 82 79 77 32℃ 84 82 81 78 75 35℃ 82 79 76 73 70 37℃ 78 75 72 68 64

[0246] Results analysis:

[0247] Considering both shelf life and viable cell rate, the optimal combination is 32℃ × 48h (shelf life 18 days, viable cell rate 81%). The results of 30℃ × 48h (shelf life 17 days, viable cell rate 82%) and 32℃ × 36h (shelf life 17 days, viable cell rate 82%) are also very close. This defines 30–33℃ × 36–48h as the optimal process window.

[0248] Fermentation at 28 ℃ was slow, inhibiting the metabolic activity of lactic acid bacteria. The slow initial acid production led to insufficient initial antibacterial activity and a short shelf life (≤15 days). At 37 ℃, the temperature was too high. Although acid production was rapid, it exerted additional heat stress on Euglena cells, significantly reducing cell viability. After 72 hours of fermentation, cell viability decreased to 64% (37 ℃) and 75% (32 ℃). The prolonged fermentation (72 hours) resulted in varying degrees of cell viability reduction at all temperature conditions, suggesting that after 60 hours of fermentation, the accumulation of metabolic byproducts began to adversely affect Euglena.

[0249] The results validate the scientific basis for the claim that "fermentation is carried out at 30–33°C for 36–48 hours".

[0250] Example 12 (Optimization and Validation of Inoculation Volume)

[0251] This embodiment verifies the effect of different probiotic inoculation amounts on preservation effect.

[0252] The steps are the same as in Example 1, except that the inoculation amount of the compound seed solution is 1%, 2%, 3%, 5%, 8%, 10%, and 15% (v / v), respectively. Each group has three replicates.

[0253] Table 12. Results of the inoculum size optimization experiment in Example 12

[0254] 1% 11 80 <![CDATA[5.2×10 6 ]]> 6.5 Insufficient initial bacterial count, slow startup 2% 14 80 <![CDATA[1.1×10 7 ]]> 7.2 The effect was acceptable but not optimal. 3% 16 81 <![CDATA[1.8×10 7 ]]> 8.2 Good results 5% 18 81 <![CDATA[2.8×10 7 ]]> 8.5 Optimal solution 8% 17 79 <![CDATA[4.5×10 7 ]]> 8.3 Good results 10% 16 78 <![CDATA[5.8×10 7 ]]> 7.8 High bacterial count intensifies nutrient competition. 15% 14 75 <![CDATA[8.2×10 7 ]]> 7.0 Over-inoculation leads to excessively rapid accumulation of products.

[0255] Results analysis:

[0256] (1) Inoculation amount is too low (<2%): initial bacterial count is only 5.2×10 6 It requires a long lag period to reach the bacterial density needed for effective antibacterial activity, and has a shelf life of only 11 days.

[0257] (2) Optimal range (3%–8%): Inoculation amounts of 3%, 5%, and 8% all achieved the goal of a shelf life of ≥16 days and a viable cell rate of ≥79%. Among them, 5% showed the best effect, with a probiotic viable cell count of 2.8 × 10⁻⁶ in 12 hours. 7 CFU / mL, at optimal equilibrium. 3% is effective but has a slightly shorter shelf life; 8% is effective.

[0258] (3) Inoculation amount is too high (>10%): The initial viable count is as high as 5.8×10 7 ~8.2×10 7 At CFU / mL, under conditions of limited carbon source (10 g / L glucose), competition for carbon sources and micronutrients intensified among probiotics and between probiotics and Euglena. This resulted in a decrease in both shelf life and viable cell rate; at a 15% inoculum, the viable cell rate dropped to 75%, and the shelf life decreased to 14 days.

[0259] This result verifies the scientific basis for the claim of "inoculation at a volume ratio of 3% to 8%".

[0260] To systematically verify the necessity and inventiveness of the technical solution of this invention, the following comparative examples are provided. Comparison of the overall effects of Example 1 and the comparative examples:

[0261] Comparative Example 1 (Euglena treated according to method CN120322157A)

[0262] Following the method disclosed in CN120322157A, Euglena were fermented using lactic acid bacteria with sufficient carbon source (30 g / L glucose). No active pH adjustment was performed, allowing the pH to decrease naturally. The remaining operations—preparation of Euglena sludge, preparation of probiotic seed culture (using the same 2:1 mixture of *Lactobacillus plantarum* and *Lactobacillus acidophilus* seed culture as in Example 1, with an inoculum size of 5% v / v), fermentation temperature of 32℃, fermentation time of 48 h, and post-treatment and storage conditions—were all the same as in Example 1, differing only in carbon source concentration and pH strategy.

[0263] Table 13. Detection data at various time points in Comparative Example 1

[0264] pH value 6.5 4.8 4.2 4.0 3.8 3.8 viable cell rate (%) 95 52 35 22 5 0 Sensory rating 7.0 5.5 4.8 4.0 3.0 2.0 Total bacterial count (CFU / mL) <![CDATA[2.5×10 7 ]]> <![CDATA[8.5×10 8 ]]> <![CDATA[1.2×10 8 ]]> <![CDATA[1.2×10 7 ]]> <![CDATA[5.6×10 6 ]]> <![CDATA[3.2×10 6 ]]> Hexanal residue (μg / kg) 125 68 48 35 42 55

[0265] Shelf life determination:

[0266] On day 3, the sensory score was 4.0 (<6 points) and the viable cell rate was 22% (<70%), therefore the shelf life was determined to be 2 days (according to the standard, day N-1). If only microbiological indicators are considered (total bacterial count <10),... 5 (Limited to CFU / mL), because the probiotics in the system are included in the total bacterial count, this indicator cannot be used alone for judgment. Considering both sensory evaluation and viable cell rate, the shelf life is extremely short.

[0267] Table 14. Comparison of key indicators between Comparative Example 1 and Example 1

[0268] carbon source concentration 10 g / L 30 g / L Comparative Example 1 used three times the carbon source of Example 1. pH strategy Active narrow-range regulation (May 8 - June 2) No regulation Comparative Example 1 naturally decreased to 3.8-4.0. Shelf life 18 days 2 days Example 1 is 9 times that of Comparative Example 1 Endpoint viable cell rate 81% (Day 18) 0% (Day 7) Comparative Example 1: Complete Cell Death End-point sensory rating 8.5 2.0 (Corruption) Comparative Example 1 is completely unacceptable Hexanal residue <2.0 μg / kg 35→55 μg / kg The rebound in the later stage of Comparative Example 1 was due to cell lysis.

[0269] Mechanism analysis:

[0270] Comparative Example 1 directly verified the complete failure of conventional lactic acid bacteria fermentation methods (represented by CN120322157A) for the preservation of live Euglena. The failure mechanism is a chain reaction of rapid acid production driven by high carbon sources, which leads to pH exceeding the tolerance limit of Euglena, resulting in acid damage, and ultimately cell death and autolysis.

[0271] Specifically, 30 g / L glucose provided the lactic acid bacteria with ample energy substrate, enabling them to undergo glycolysis and lactic acid fermentation at an unrestricted rate. After 24 hours of fermentation, the pH dropped to 4.2, and more than half of the Euglena cells had lost viability (52% viability). By the end of fermentation at 48 hours, the pH stabilized at 3.8-4.0, with only 22% viability. Under this highly acidic environment, the protein outer membrane of Euglena underwent irreversible denaturation, leading to the leakage of intracellular contents. In the later stages of storage (days 5-7), the hexanal content actually increased (42→55 μg / kg), further evidence of cell lysis and the release of contents.

[0272] A comparison with Example 1 reveals the essential innovation of this invention. Comparative Example 1 represents the traditional fermentation approach of "sacrificing algal cells for antibacterial purposes," while Example 1 achieves both antibacterial and cell preservation through low-carbon source restriction and active, precise pH control. The difference between the two is not merely a quantitative change in parameters, but a qualitative leap in the technical approach. Comparative Example 1 demonstrates that without the low-carbon source combined with narrow-range pH control design of this invention, even using the same fermentation strains and temperature, it is impossible to achieve live Euglena preservation.

[0273] Comparative Example 2 (using microalgae with cell walls as the subject of the method of the present invention)

[0274] Chlorella vulgaris was used instead of Euglena, and the rest of the operation was exactly the same as in Example 1 (low carbon source 10 g / L glucose, pH actively adjusted to 5.8-6.2, mixed seed liquid 2:1, 5% v / v inoculation, fermentation at 32℃ for 48h).

[0275] Table 15. Comparison of effects between Comparative Example 2 and Example 1

[0276] Processing object characteristics No cell wall, only a protein outer membrane It has a tough cellulose cell wall Initial viable cell rate (%) 96 95 Shelf life (days) 18 21 Endpoint viable cell rate (%) 81 90 End-point sensory rating 8.5 6.5 Hexanal residue (μg / kg) <2.0 85 2-Octenal residue (μg / kg) <1.0 52 1-Octen-3-ol residue (μg / kg) <1.5 38 Evaluation of deodorization effect Completely eliminate the fishy smell of algae A significant amount of fishy-smelling substances remained.

[0277] Mechanism analysis:

[0278] Comparative Example 2 reveals a scientifically significant discovery: the deodorizing effect of the method of the present invention is specific to algae species, which is due to the presence or absence of cell wall structure.

[0279] Chlorella has a tough cellulose / hemicellulose cell wall (about 20-30 nm thick) that acts as a molecular sieve, allowing small molecules of nutrients to pass through but blocking the penetration of large molecules of proteins and enzymes.

[0280] Under the low carbon source (10 g / L) and mild pH (5.8-6.2) conditions specified in this invention, the probiotics did not undergo the explosive metabolism characteristic of sugar-added fermentation. Consequently, their secreted aldehyde dehydrogenase (ALDH, molecular weight approximately 55 kDa) and other extracellular enzymes could not effectively penetrate the cell wall barrier of *Chlorella vulgaris*. Therefore, although *Chlorella vulgaris* cells also contain odor-causing substances such as hexanal and 2-octenal (initial levels similar to *Euglena*), these substrates were confined to the inner side of the cell wall and could not fully react with the probiotic ALDH on the outer side of the cell wall, resulting in extremely poor deodorization (hexanal residue as high as 85 μg / kg).

[0281] In contrast, Euglena lacks a cell wall and is enclosed only by a protein pellicle. This pellicle is composed of cysteine-rich protein bands, and its structure is elastic, allowing small and medium-molecular-weight proteins to pass freely. Therefore, the odorous aldehydes within the Euglena cells can freely diffuse to the extracellular space to contact the probiotic ALDH, while ALDH can also partially permeate into the periplasmic space of the Euglena cells to exert its catalytic activity, achieving a highly efficient match between substrate and enzyme. This structural difference explains the fundamental reason why Comparative Example 2 failed to remove the odor while Example 1 succeeded.

[0282] Furthermore, Comparative Example 2 showed a longer shelf life (21 days) and a higher cell viability (90%) than Example 1 (18 days and 81%), further demonstrating that Euglena is more sensitive to the metabolic environment due to the lack of cell wall protection. Chlorella, on the other hand, has a stronger tolerance to pH fluctuations and metabolic products due to the protection of its cell wall, which also explains why the technical difficulty of preserving Euglena alive is higher than that of general microalgae.

[0283] This comparative example also indirectly proves that the technical solution of this invention is specifically designed for the physiological structural characteristics of Euglena (no cell wall, sensitive to the external environment, but allowing free permeability of enzymes and substrates), and cannot be simply extended from other microalgae species. This invention fully utilizes the structural characteristics of Euglena—its fragile protective nature and convenient permeability—by limiting the acid production rate with a low carbon source (protecting cell-wall-less Euglena from acid damage) and narrow-range pH control (creating an optimal enzyme-substrate reaction environment), achieving a balance between selective antibacterial activity and efficient deodorization.

[0284] Comparative Example 3 (only buffer salts and carbon sources were added, without inoculation with probiotics)

[0285] The procedure is the same as in Example 1, except that no probiotics are inoculated. In the same manner as in Example 1, 10 g / L glucose and 0.1 mol / L phosphate buffer (pH 6.5) are added, and the mixture is stirred at 32°C and 100 rpm for 48 hours, but no bacterial strains are introduced. Subsequently, Euglena biomass is collected by centrifugation at 4000 rpm for 10 min and refrigerated at 4°C.

[0286] Table 16. Detection data at various time points in Comparative Example 3

[0287] pH value 6.4 6.3 6.2 6.0 viable cell rate (%) 96 80 72 58 Sensory rating 7.0 5.5 4.5 3.8 Total bacterial count (CFU / mL) 8.5×10³ <![CDATA[5.2×10 5 ]]> <![CDATA[2.8×10 6 ]]> <![CDATA[1.5×10 7 ]]> Hexanal residue (μg / kg) 128 115 98 105 2-Octenal residue (μg / kg) 90 82 72 78

[0288] Shelf life determination:

[0289] The total bacterial count on day 3 was 5.2 × 10⁻⁶. 5 CFU / mL (>10) 5 The concentration of CFU / mL was 5.5 (<6 points), therefore the shelf life was determined to be 2 days.

[0290] Table 17. Comparison of key indicators between Comparative Example 3 and Example 1

[0291] Probiotic inoculation Compound (2:1), 5% v / v none — Shelf life 18 days 2 days Example 1 is 9 times that of Comparative Example 3. Endpoint viable cell rate 81% (Day 18) 58% (Day 7) Comparative Example 3: Continuous Cell Death Total bacterial count (day 3) <![CDATA[2.8×10 7 CFU / mL]]> <![CDATA[5.2×10 5 CFU / mL]]> Comparative Example 3 showed excessive levels of miscellaneous bacteria, while Example 1 showed probiotics. Hexanal residue <2.0 μg / kg 98-128 μg / kg Comparative Example 3 showed no effect in removing fishy odor. 2-Octenal residue <1.0 μg / kg 72-90 μg / kg Comparative Example 3: No reduction in fishy odor substances

[0292] Mechanism analysis:

[0293] Comparative Example 3 confirms the irreplaceable role of probiotics in the Euglena preservation system in occupying ecological niches and producing antibacterial active substances.

[0294] Even without probiotic inoculation, the same low-carbon source (10 g / L glucose) and buffer salt system (0.1 mol / L phosphate) as in Example 1 were added, but these nutrients instead became a culture medium for the proliferation of putrefactive bacteria. On day 0, the total bacterial count was only 8.5 × 10³ CFU / mL, but due to the lack of competitive exclusion from probiotics, the sensory score subsequently dropped sharply from 7.0 to 5.5, and the viable cell percentage decreased from 96% to 80%. By day 7, the total bacterial count had reached 1.5 × 10³ CFU / mL. 7The CFU / mL concentration further decreased to 58% and the sensory score was 3.8.

[0295] The content of hexanal remained at a relatively high level of 98-128 μg / kg and remained basically unchanged, while the content of 2-octenal also remained at a high level of 72-90 μg / kg, which verified that the buffer salt itself does not have the function of deodorizing.

[0296] This comparative example powerfully demonstrates that the preservation effect of the present invention is not a simple physicochemical protection of the buffer salt system, but rather relies on a complete biological preservation mode in which probiotics first establish a dominant ecological niche in the system.

[0297] Comparative Example 4 (inoculation with compound probiotics + active pH regulation, but without the addition of a carbon source)

[0298] The steps are the same as in Example 1, except that glucose is not added. A seed culture of *Lactobacillus plantarum* and *Lactobacillus acidophilus* in a 2:1 ratio is inoculated at 5% v / v and fermented at 32°C for 48 hours. The pH is actively adjusted between 5.8 and 6.2 throughout the process. Post-treatment is the same as in Example 1.

[0299] Table 18. Detection data at various time points in Comparative Example 4

[0300] pH value (after adjustment) 6.4 6.2 6.0 5.9 5.8 5.8 viable cell rate (%) 95 88 85 82 78 68 Sensory rating 7.0 7.0 6.8 6.5 6.0 5.5 Hexanal residue (μg / kg) 125 88 62 48 45 52 2-Octenal residue (μg / kg) 85 55 38 28 30 35 48-hour probiotic live bacteria count (CFU / mL) <![CDATA[2.5×10 7 ]]> <![CDATA[1.2×10 7 ]]> <![CDATA[8×10 6 ]]> — — — ALDH enzyme activity (48h, U / mg protein) — — 0.28 — — —

[0301] Shelf life determination:

[0302] On day 8, the sensory score was 6.0 (the critical value), and on day 10 it dropped to 5.5 (<6 points). The viable cell rate dropped to 68% (<70%), so the shelf life was determined to be 8 days.

[0303] Table 19. Comparison of key indicators between Comparative Example 4 and Example 1

[0304] carbon source concentration 10 g / L glucose 0 g / L Comparative Example 4: Carbon source completely absent Shelf life 18 days 8 days Example 1 is 2.25 times that of Comparative Example 4. 48h probiotic live bacteria count <![CDATA[3.0×10 8 CFU / mL]]> <![CDATA[8×10 6 CFU / mL]]> Comparative Example 4 is only 2.7% of Example 1. ALDH enzyme activity (48h) 0.45 U / mg 0.28 U / mg Comparative Example 4 is only 62% of Example 1. End-point sensory rating 8.5 (Day 18) 5.5 (Day 10) Comparative Example 4 showed poor deodorizing and antibacterial effects. Hexanal residue <2.0 μg / kg 45-52 μg / kg Comparative Example 4 was severely inadequate in removing fishy odor.

[0305] Mechanism analysis:

[0306] Comparative Example 4 reveals the fundamental impact of carbon source supply on the preservation function of probiotics.

[0307] Without the addition of an exogenous carbon source, the organic substrates available to probiotics are limited to trace amounts of organic matter secreted extracellularly by Euglena during photosynthesis or basal metabolism. The viable bacterial count increased from an initial 2.5 × 10⁻⁶ at 48 hours. 7 CFU / mL decreased to 8×10 6 The CFU / mL (only 2.7% of that in Example 1) indicates that the probiotics not only failed to proliferate, but also gradually died due to carbon source depletion.

[0308] The ALDH enzyme activity was only 0.28 U / mg protein (62% of Example 1), which greatly reduced the deodorizing ability. The residual amounts of hexanal and 2-octenal were only partially reduced (125→45 μg / kg and 85→30 μg / kg), which was far from achieving a complete deodorizing effect.

[0309] Comparative Example 5 (inoculated with compound probiotics + low carbon source (10 g / L), but without active pH regulation)

[0310] The procedure was the same as in Example 1, except that no pH adjustment was performed. Glucose 10 g / L was used as a 2:1 mixture of seed culture and inoculated at 5% v / v. Fermentation was carried out at 32°C for 48 hours. No pH intervention was performed during fermentation; pH was allowed to change naturally. Post-treatment was the same as in Example 1.

[0311] Table 20. Detection data at various time points in Comparative Example 5

[0312] pH value 6.4 5.6 5.2 4.8 4.5 4.2 viable cell rate (%) 95 88 78 65 52 38 Sensory rating 7.0 7.2 6.8 6.2 5.8 5.0 Hexanal residue (μg / kg) 126 42 28 18 22 25 2-Octenal residue (μg / kg) 88 25 15 10 12 15 48h ALDH enzyme activity (U / mg protein) — 0.38 0.35 0.30 0.25 —

[0313] Shelf life determination:

[0314] On day 6, the viable cell rate was 65% (<70%), and on day 8, the sensory score was 5.8 (<6 points). Therefore, the shelf life was determined to be 6 days.

[0315] Table 21. Comparison of key indicators between Comparative Example 5 and Example 1

[0316] pH strategy Active regulation 5.8-6.2 No regulation Core variables Fermentation endpoint pH 5.8-6.2 (after adjustment) 4.8 (Nature) Comparative Example 5 broke through the tolerance limit of Euglena. Storage endpoint pH May 8 - June 2 (Day 18) 4.2 (Day 10) pH continues to decrease Shelf life 18 days 6 days Example 1 is 3 times that of Comparative Example 5. Endpoint viable cell rate 81% (Day 18) 38% (Day 10) Comparative Example 5: Cells severely damaged ALDH enzyme activity (48h) 0.45 U / mg 0.35 U / mg pH decrease inhibits ALDH activity Hexanal residue <2.0 μg / kg 18→25 μg / kg (rebound) Comparative Example 5 shows a mid-to-late stage rebound.

[0317] Mechanism analysis:

[0318] Comparative Example 5 is the core evidence revealing the coupling dependence between low carbon sources and active pH regulation.

[0319] Although the carbon source concentration in Comparative Example 5 was 10 g / L (the same as in Example 1, within the preferred range of 8-15 g / L), the probiotics continuously produced acid, driving the system pH down due to the lack of any pH adjustment. During fermentation, the pH dropped to 5.5 at 24 h, 5.0 at 48 h, and 4.8 at 60 h. During storage, the pH further decreased to 4.2 on day 10.

[0320] The trajectory of cell viability precisely reveals the quantitative relationship between pH and cell damage. On day 2, at pH 5.6, the cell viability was 88%, which was basically normal; on day 4, at pH 5.2, it dropped to 78%, showing a noticeable decline; on day 6, at pH 4.8 (below the critical value of 5.0), it plummeted to 65%, below the 70% preservation standard; on day 10, at pH 4.2, only 38% remained, indicating that the cells had suffered severe and irreversible acidic damage.

[0321] ALDH enzyme activity decreased continuously with decreasing pH. It dropped from 0.38 U / mg on day 2 (pH 5.6) to 0.25 U / mg on day 8 (pH 4.5). This is because the optimal pH for ALDH is typically between 7.5 and 8.5. A pH below 5.0 leads to protonation of histidine and cysteine ​​residues in the enzyme's catalytic center, altering the enzyme's three-dimensional conformation and coenzyme binding capacity, resulting in a significant loss of catalytic activity.

[0322] The residual hexanal level decreased to 18 μg / kg during the middle of storage (day 6, pH 4.8). Subsequently, due to a further decrease in ALDH activity and the release of intracellular hexanal from the death and lysis of some Euglena cells, the hexanal content rebounded to 22-25 μg / kg in the later stage of storage. 2-Octenal also showed a similar trend of first decreasing and then increasing (88→10→15 μg / kg). This is precisely the significant difference between the system without active pH regulation and the system in Example 1, which maintained a precise pH of 5.8-6.2 throughout the entire process. In Example 1, the pH was always kept within the optimal working conditions for ALDH, ensuring the long-lasting stability of the deodorizing effect.

[0323] More importantly, the comparison of the results of Comparative Example 5 with Comparative Example 4 and Example 1 reveals the inseparable coupling relationship between low carbon source and active pH regulation.

[0324] Comparative Example 4 (No carbon source + pH control): Shelf life 8 days. This proves that without carbon source supply and pH control alone, probiotics cannot effectively proliferate and function, resulting in preservation failure.

[0325] Comparative Example 5 (with low carbon source + no pH control): Shelf life 6 days. This demonstrates that having only a low carbon source without pH control leads to acid accumulation that directly damages Euglena cells and inhibits deodorizing enzyme activity, resulting in preservation failure.

[0326] Example 1 (Low carbon source + active pH control): Shelf life 18 days. This demonstrates that the desired effect can only be achieved when both conditions are met simultaneously.

[0327] This comparative system fully illustrates that low-carbon sources provide the possibility and time for precise pH control by limiting the rate of acid production (slowing down the rate at which pH drops below 5.8), while precise pH control compensates for the potential residual acid damage risk in low-carbon source strategies. Both play their respective roles, are mutually dependent, and neither can be dispensed with.

[0328] Comparative Example 6 (inoculation with compound probiotics + active pH regulation, but using a high carbon source (40 g / L))

[0329] The steps are the same as in Example 1, except that the amount of glucose added is 40 g / L. The other conditions (2:1 mixed seed culture, 5% v / v inoculation, fermentation at 32℃ for 48 h, active pH adjustment throughout the process from 5.8 to 6.2, post-treatment and cold storage) are completely consistent with Example 1.

[0330] Table 22. Detection data at various time points in Comparative Example 6

[0331] pH value (after adjustment) 6.4 6.0 5.9 5.8 5.8 pH adjustment frequency (times / 48h) — 8 9 7 6 viable cell rate (%) 95 75 65 58 50 Sensory rating 7.0 7.0 6.5 6.2 5.8 Hexanal residue (μg / kg) 124 32 22 15 18 2-Octenal residue (μg / kg) 88 18 12 8 10 ALDH enzyme activity (48h, U / mg protein) — 0.25 0.20 0.18 —

[0332] Shelf life determination:

[0333] On day 9, the sensory score was 6.2 (borderline), and on day 12 it dropped to 5.8 (<6 points). The viable cell rate of 50% was far below the 70% standard, so the shelf life was determined to be 9 days.

[0334] Table 23 Comparison of key indicators between Comparative Example 6 and Example 1

[0335] carbon source concentration 10 g / L 40 g / L Comparative Example 6 used a carbon source that was 4 times that of Example 1. Shelf life 18 days 9 days Example 1 is twice that of Comparative Example 6. Endpoint viable cell rate 81% (Day 18) 50% (Day 12) Comparative Example 6: Cell viability halved ALDH enzyme activity (48h) 0.45 U / mg 0.18 U / mg Comparative Example 6 is only 40% of Example 1 pH adjustment frequency 3 times / 48h 8-9 times / 48h The difficulty of regulation increases sharply with the ratio of 6. Hexanal residue <2.0 μg / kg 15-18 μg / kg Comparative example 6 showed poor deodorization effect. 2-Octenal residue <1.0 μg / kg 8-10 μg / kg Comparative example 6 showed poor deodorization effect.

[0336] Mechanism analysis:

[0337] Comparative Example 6 reveals the important conclusion that even with active pH regulation, the preservation effect still deteriorates significantly if the carbon source concentration exceeds the appropriate range, providing direct experimental evidence for setting the upper limit of carbon source concentration (15 g / L) in the claims.

[0338] Under conditions of a high carbon source of 40 g / L, although the same active pH control strategy (target range 5.8-6.2) as in Example 1 was employed, the high concentration of glucose led to excessively rapid acid production, drastically increasing the difficulty of pH control and severely reducing process operability. The rapid increase in the acid production rate caused a surge in the frequency of pH drops below 5.8, requiring control 8-9 times per 48 hours, compared to only 3 times per 48 hours in Example 1. In actual operation, such frequent addition of alkali results in an extremely narrow process window, and even a slight delay (such as uneven mixing of the alkali solution) can cause the pH to exceed the lower limit. This significantly reduces the industrial operability and scale-up feasibility of the technical solution.

[0339] The viable cell rate dropped to 75% on day 3, 65% on day 6, and only 50% by day 12. This loss of cell viability cannot be avoided by pH regulation alone and is a direct consequence of hyperosmolar physical stress.

[0340] High carbon sources significantly inhibited ALDH expression through carbon metabolite repression. In Comparative Example 6, the ALDH enzyme activity after 48 hours was only 0.18 U / mg protein, which is 40% of that in Example 1 (0.45 U / mg). Hexanal residues were 15-18 μg / kg, and 2-octenal residues were 8-10 μg / kg, which are more than 7.5 times and 8 times higher than those in Example 1, respectively.

[0341] By combining the ALDH enzyme activity data from Comparative Example 4 (no carbon source, ALDH 0.28 U / mg), Example 1 (low carbon source 10 g / L, ALDH 0.45 U / mg), and Comparative Example 6 (high carbon source 40 g / L, ALDH 0.18 U / mg), a carbon source concentration-enzyme activity curve was constructed. The ALDH enzyme activity corresponding to carbon source concentrations of 0→10→40 g / L increased from 0.28 to 0.45 to 0.18 U / mg, showing a relationship of initial increase followed by decrease. This curve provides quantitative evidence for the scientific rationale of the 8-15 g / L carbon source range in the claims. This range falls precisely in the rising segment and peak region of the bell curve, representing the concentration range where the inducing effect of low carbon source and the repressive effect of carbon metabolites reach optimal balance.

[0342] Furthermore, the comparison between Comparative Example 6 and Comparative Example 4 revealed that too little carbon source (0 g / L) resulted in insufficient probiotic metabolic activity (shelf life of 8 days), while too much carbon source (40 g / L) led to excessive and uncontrolled metabolism (shelf life of 9 days). Only 10 g / L achieved the optimal balance for preservation. This further confirms the non-obviousness of the low carbon source concentration selected in Example 1; it was not simply chosen arbitrarily from a broad range, but was precisely determined based on a systematic study of Euglena tolerance thresholds, probiotic metabolic requirements, and enzymatic response curves.

[0343] Comparative Example 7 (using only Lactobacillus plantarum + low carbon source (10 g / L) + pH adjustment)

[0344] The procedure is the same as in Example 1, except that only *Lactiplantibacillus plantarum* is used, with an inoculum size of 5% v / v, the same as the total inoculum size of the compound. The carbon source is 10 g / L glucose, and the pH is actively adjusted from 5.8 to 6.2 throughout the process. Fermentation is carried out at 32°C for 48 hours.

[0345] Table 24. Detection data at various time points in Comparative Example 7

[0346] pH value (after adjustment) 6.4 6.2 5.9 5.8 5.8 5.8 viable cell rate (%) 95 88 82 78 75 74 Sensory rating 7.0 7.5 7.2 7.0 6.8 6.5 Hexanal residue (μg / kg) 125 28 18 12 15 18 2-Octenal residue (μg / kg) 86 18 10 6 8 10 48h ALDH enzyme activity (U / mg protein) — 0.35 — — — —

[0347] Shelf life determination:

[0348] On day 12, the sensory score was 6.8 (>6 points), and the viable cell rate was 75% (>70%). On day 13, the sensory score was 6.5 (>6 points), and the viable cell rate was 74% (>70%). Both the sensory score and viable cell rate decreased to the threshold on day 13. Monitoring was extended to day 14, and the sensory score dropped to 5.8 (<6 points), therefore the shelf life was determined to be 13 days.

[0349] Table 25. Comparison of key indicators between Comparative Example 7 and Example 1

[0350] strains Compound (2:1) Lactobacillus plantarum only Core variables Shelf life 18 days 13 days Example 1 Comparison Example 7 (5 days) Endpoint viable cell rate 81% (Day 18) 74% (Day 13) The ratio is slightly lower than 7. ALDH enzyme activity (48h) 0.45 U / mg 0.35 U / mg Comparative Example 7 is 78% of Example 1. Hexanal residue <2.0 μg / kg 12→18 μg / kg (later rebound) Comparative Example 7: Incomplete removal of fishy smell

[0351] Mechanism analysis:

[0352] Comparative Example 7 accurately reveals the shortcomings of a single Lactobacillus plantarum in the initial acid production initiation speed, as well as the differences in metabolic division of labor in the time dimension compared with the compound scheme in Example 1.

[0353] After 12 hours of fermentation, *Lactobacillus plantarum* showed a significantly slower acid production initiation rate compared to the compound formulation containing *Lactobacillus acidophilus*. This is because the activation rate of *Lactobacillus plantarum*' glucose transport system is low in the early stages of fermentation, resulting in a low glycolysis flux. This characteristic means that the inhibition window against contaminating microorganisms was not fully opened in the first 24 hours of fermentation. Although no explosive growth of contaminating microorganisms occurred with the assistance of active pH regulation (5.8-6.2), the consequences of incomplete initial inhibition gradually became apparent in the later stages of storage (days 9-14). The remaining small number of contaminating microorganisms gradually resumed proliferation, and the sensory score continuously decreased from 7.0 to 5.8, while the viable cell rate also decreased from 78% to 74%. The combined defects of insufficient acid inhibition in the early stage and the inability to provide long-term antibacterial effects in the later stage in Comparative Example 7 shortened its shelf life by 5 days compared to Example 1.

[0354] Comparative Example 8 (using only Lactobacillus acidophilus + low carbon source (10 g / L) + pH adjustment)

[0355] The procedure is the same as in Example 1, except that only Lactobacillus acidophilus (inoculation amount is the same as the total inoculation amount of the compound, 5% v / v) is used. The carbon source is 10 g / L glucose, the pH is actively adjusted to 5.8-6.2 throughout the process, and fermentation is carried out at 32℃ for 48 h.

[0356] Table 26. Detection data at various time points in Comparative Example 8

[0357] pH value (after adjustment) 6.4 6.0 5.8 5.8 5.8 viable cell rate (%) 95 86 78 73 71 Sensory rating 7.0 7.2 7.0 6.8 6.5 Hexanal residue (μg / kg) 126 35 22 16 18 2-Octenal residue (μg / kg) 85 20 12 8 10 48h ALDH enzyme activity (U / mg protein) — 0.28 — — —

[0358] Shelf life determination:

[0359] On day 12, the sensory score was 6.5 (>6 points), and the viable cell rate was 71% (>70%, borderline level). On day 13, the sensory score dropped to 5.6 (<6 points), therefore the shelf life was determined to be 12 days.

[0360] Table 27. Comparison of key indicators between Comparative Example 8 and Example 1

[0361] strains Compound (2:1) Lactobacillus acidophilus only Core variables Shelf life 18 days 12 days Example 1 Comparison with Example 8 (6 days) Endpoint viable cell rate 81% (Day 18) 71% (Day 12) The ratio of 8 is too low. ALDH enzyme activity (48h) 0.45 U / mg 0.28 U / mg Comparative Example 8 is only 62% of Example 1. Hexanal residue <2.0 μg / kg 16-18 μg / kg Comparative ratio 8 showed poor deodorization effect.

[0362] Mechanism analysis:

[0363] Comparative Example 8 precisely revealed the serious shortcomings of Lactobacillus acidophilus alone in the long-term antibacterial effect in the later stage, and served as a control with Comparative Example 7.

[0364] Lactobacillus acidophilus initiates acid production extremely rapidly, maintaining a sensory score above 7.0 and a viable cell rate above 78% during the early storage period (days 0-6). As the sensory score drops from 7.0 to 6.5 and the viable cell rate decreases from 78% to 71%, the preservation barrier gradually collapses.

[0365] Furthermore, the ALDH enzyme activity of Comparative Example 8 was only 0.28 U / mg (62% of that in Example 1), with hexanal residues of 16-18 μg / kg and 2-octenal residues of 8-10 μg / kg. Its deodorizing effect was the worst among Comparative Examples 7 and 8, as well as Example 1. This may be related to the differences in the number and expression regulation of aldehyde dehydrogenase homologs in the Lactobacillus acidophilus genome compared to Lactobacillus plantarum.

[0366] Table 28. Comparison of complementary bacterial strains in Comparative Examples 7, 8 and Example 1

[0367] Initial acid production start-up slow quick Moderate ALDH enzyme activity Medium (0.35 U / mg) Low (0.28 U / mg) High (0.45 U / mg) Shelf life 13 days 12 days 18 days core defects Slow initial antibacterial action The long-term defense line collapsed in the later stages. None, fully functional

[0368] Mechanism summary:

[0369] Comparative Examples 7 and 8 formed a precise mirror comparison, jointly verifying the scientific validity and non-obviousness of the "2:1 dual-strain compound" of this invention. The weakness of Comparative Example 7 was its slow initial growth, while that of Comparative Example 8 was its weaker growth in the later stages. The effective shelf life of each strain when used alone was 13 days and 12 days, respectively, both significantly different from the 18 days in Example 1 (P < 0.05). Example 1, through a 2:1 compound, seamlessly integrated the advantages of *Lactobacillus acidophilus* and *Lactobacillus plantarum* over time, forming a complete metabolic relay. The ALDH enzyme activity when both coexisted (0.45 U / mg) was significantly higher than that when either was used alone (0.35 U / mg and 0.28 U / mg), revealing a possible synergistic enzyme activity induction effect between the two, further improving the deodorization efficiency. This "1 + 1 > 2" effect of the compound system is unattainable by single-strain solutions.

[0370] Summary of the effects of Example 1 and all comparative examples, and synergistic effect analysis:

[0371] Table 29. Overall Comparison of the Results of Example 1 and All Comparative Examples

[0372] Example 1 10 Active regulation 5.8-6.2 2:1 blend 18 81 (Day 18) 8.5 <2.0 0.45 Comparative Example 1 30 No regulation 2:1 blend 2 0 (Day 7) 3.0 (Day 5) 35→55 — Comparative Example 2 10 Active regulation 5.8-6.2 2:1 blend 21 (Chlorella) 90 (Chlorella) 6.5 85 — Comparative Example 3 10 No fermentation none 2 58 (Day 7) 3.8 (Day 7) 98-128 — Comparative Example 4 0 Active regulation 5.8-6.2 2:1 blend 8 68 (Day 10) 5.5 (Day 10) 45-62 0.28 Comparative Example 5 10 No regulation 2:1 blend 6 38 (Day 10) 5.0 (Day 10) 18→25 0.35 Comparative Example 6 40 Active regulation 5.8-6.2 2:1 blend 9 50 (Day 12) 5.8 (Day 12) 15-18 0.18 Comparative Example 7 10 Active regulation 5.8-6.2 Lactobacillus plantarum only 13 74 (Day 13) 6.5 (Day 13) 12-18 0.35 Comparative Example 8 10 Active regulation 5.8-6.2 Lactobacillus acidophilus only 12 71 (Day 12) 6.5 (Day 12) 16-18 0.28

[0373] The following key conclusions can be drawn from the data system in Table 29:

[0374] (1) Each technical feature is irreplaceable, and the absence or deviation of any feature will lead to a significant deterioration in performance.

[0375] The consequences of lacking the "low carbon source" characteristic: Comparative Example 4 (0 g / L carbon source) had a shelf life of 8 days; Comparative Example 6 (40 g / L carbon source) had a shelf life of 9 days. The probiotics in the former were unable to function due to the lack of an energy source (the number of viable bacteria after 48 hours was only 2.7% of that in Example 1), while the probiotics in the latter suffered from excessive metabolism and enzyme inhibition (ALDH enzyme activity was only 40% of that in Example 1). The shelf life of both was less than half that of Example 1.

[0376] The consequences of lacking the "narrow-range pH control" feature: Comparative Example 1 (high carbon source + no pH adjustment, referring to existing technology CN120322157A) had a shelf life of only 2 days, and all Euglena died within 7 days, proving that the conventional fermentation method was completely ineffective; Comparative Example 5 (low carbon source but no pH adjustment) had a shelf life of 6 days, and the pH naturally dropped to 4.2, with only 38% of cells remaining viable. Comparative Example 5 demonstrates that even with a suitable carbon source concentration, abandoning pH control still leads to uncontrolled acidity in the system, resulting in preservation failure.

[0377] The consequences of lacking the "dual-strain combination" feature: Comparative Example 7 (Lactobacillus plantarum only) had a shelf life of 13 days, with its core weakness being slow initial acid production initiation; Comparative Example 8 (Lactobacillus acidophilus only) had a shelf life of 12 days, with its core weakness being the collapse of the long-term antibacterial defense in the later stages. Single strains have structural defects in terms of "initial acid production initiation" or "long-term antibacterial effect in the later stages," and the functions of the two cannot form an effective connection in the time dimension.

[0378] (2) Existing technologies are completely ineffective against Euglena. Comparative Example 1, represented by CN120322157A, uses a conventional lactic acid bacteria fermentation method (high carbon source 30 g / L + no pH adjustment). After 24 hours of fermentation, the pH drops to 4.0, the cell viability is only 22%, and the shelf life is only 2 days, showing no value for preserving Euglena alive. Comparative Example 3 demonstrates that without the bioprotective effect of probiotics, the buffer salts and carbon source instead become a natural growth medium for miscellaneous bacteria, resulting in a shelf life of only 2 days as well.

[0379] (3) The effect of this invention stems from the synergistic combination of three technical features at a specific level, exhibiting non-additive properties. The 18-day shelf life of Example 1 is significantly better than any single-factor missing or deviated group, and is 5 days longer than the optimal single-factor missing group (Comparative Example 7, 13 days). Using the Jin Zhengjun Q-value method, the Q-value of the combined effect of the three features is >1.15, confirming a significant synergistic effect.

[0380] The biological basis of this synergistic effect lies in the fact that the low-carbon source, by limiting the acid production rate, provides a controllable operating window and sufficient response time for precise pH regulation. The narrow pH range (5.8-6.2) creates optimal enzymatic conditions for the efficient expression of the probiotic ALDH, while simultaneously falling within the selective balance range between inhibiting contaminating bacteria and protecting Euglena. The 2:1 dual-strain formulation, within this low-carbon source and narrow-pH environment, forms a complete metabolic relay chain. These three elements are interdependent, mutually dependent, and indispensable, jointly constructing a mild and long-lasting selective biopreservation system specifically designed for cell-wall-less Euglena.

[0381] Industrial applicability

[0382] The method of this invention operates under mild conditions, and all required reagents are commonly used raw materials in the food industry. The *Lactobacillus plantarum* and *Lactobacillus acidophilus* used are safe strains listed in the "List of Microbial Strains that Can Be Used in Food." The treated Euglena biomass exhibits superior cell viability, sensory quality, and antioxidant activity compared to existing preservation methods, and can be directly applied to the high-end functional food sector. This invention can be widely applied in Euglena cultivation enterprises and microalgae deep processing enterprises, significantly extending product shelf life, improving product quality and market competitiveness, and possessing significant industrial promotion value and economic benefits.

Claims

1. A method for preserving Euglena through fermentation using a low-carbon source and narrow-range pH regulation probiotics, characterized in that... Includes the following steps: (1) Harvest fresh Euglena, wash and concentrate to obtain fresh Euglena mud with a water content of 85% to 95%; (2) Add carbon source and buffer salt to Euglena sludge, mix evenly to obtain fermentation substrate; the carbon source is one of glucose, fructose or sucrose, with a final concentration of 8-15 g / L; the buffer salt is a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer system with a concentration of 0.05-0.15 mol / L, so that the initial pH of the fermentation substrate is 6.0-6.8; (3) Lactobacillus plantarum and Lactobacillus acidophilus were activated separately to obtain a viable count of 1×10⁻⁶. 8 ~1×10 9 CFU / mL seed culture; mix the two seed cultures at a volume ratio of 2:1 to obtain a compound probiotic seed culture; (4) Inoculate the compound probiotic seed liquid from step (3) into the fermentation substrate from step (2) at a volume ratio of 3% to 8%, and ferment at 30 to 33°C for 36 to 48 hours; during the fermentation process, the pH of the system is controlled within the range of 5.8 to 6.2 by adding alkaline solution throughout. (5) After fermentation, solid-liquid separation is carried out, the Euglena biomass is collected and stored at 4℃. The Euglena mentioned in step (1) is Euglena gracilis. The cell concentration of Euglena sludge in step (1) is 1×10⁻⁶. 4 ~1×10 6 cells / mL. The carbon source in step (2) is glucose, with a final concentration of 10 g / L. The buffer salt concentration in step (2) is 0.1 mol / L and the initial pH is 6.

5. The fermentation temperature in step (4) is 32°C, the fermentation time is 48 hours, and the inoculum amount is 5% (v / v).

2. A preserved Euglena product prepared by the method according to any one of claims 1.

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