Plant lactobacillus and its application in chlorella pyrenoidosa fermented beverage

Abstract

The application discloses a Lactiplantibacillus plantarum and application of the Lactiplantibacillus plantarum in fermentation beverage of Chlorella pyrenoidosa. In the application, a Lactiplantibacillus plantarum is screened and separated from an outdoor Chlorella pyrenoidosa cultivation pond, and is named as Lactiplantibacillus plantarum HN3. The Lactiplantibacillus plantarum HN3 has been preserved in the Guangdong Microbial Culture Collection Center, No. 59 Building, 5th Floor, Guangzhou City, on July 26, 2024, and has a preservation number of GDMCC No: 64816. The Lactiplantibacillus plantarum HN3 has good fermentation performance, acid tolerance and bile salt tolerance, can degrade bad odor substances of Chlorella pyrenoidosa liquid, produce new pleasant odor substances, and can be used as a production strain of Chlorella pyrenoidosa fermentation beverage.

Description

Technical Field

[0001] This invention belongs to the field of fermentation and food biotechnology, and specifically relates to a strain of *Lactobacillus plantarum* and its application in fermented beverages made from *Chlorella proteoglycans*. Background Technology

[0002] *Auxenochlorella pyrenoidosa*, a single-celled green alga belonging to the genus *Auxenochlorella* in the phylum Chlorophyta, is a freshwater, spherical, single-celled algae with both plant and microbial characteristics, and is widely distributed. The protein content of *Auxenochlorella pyrenoidosa* can reach 55-65% of its cell dry weight, containing 18 amino acids, of which essential amino acids account for more than 40% of the total amino acid content, exceeding 20% ​​of the cell dry weight. Its essential amino acid index (EAAI) is 1.35, higher than the recommended protein intake. *Auxenochlorella pyrenoidosa* is also rich in dietary fiber and polyunsaturated fatty acids (such as linolenic acid and linolenic acid). In addition, it contains many essential nutrients and trace elements, including vitamins B, C, D, and E, niacin, folic acid, pantothenic acid, as well as lutein, calcium, iron, and zinc. *Auxenochlorella pyrenoidosa* also possesses many physiological functions, such as cell regeneration, free radical scavenging, anti-tumor activity, promotion of antigen-specific immunity, and T-cell proliferation and activation.

[0003] Lactic acid bacteria are a group of Gram-positive, non-spore-forming bacteria that ferment carbon sources and produce lactic acid and other organic acids. They are widely used in the food fermentation industry. Commonly used lactic acid bacteria in the food industry mainly include strains of the genera *Streptococcus* and *Lactobacillus*, such as *Streptococcus thermophilus*, *Lactobacillus acidophilus*, *Lactobacillus delbrueckii* subsp. bulgaricus, *Lactobacillus casei*, *Lactobacillus rhamnosus*, *Lactobacillus helveticus*, and *Lactobacillus plantarum*. These strains are generally considered to be GRAS (Generally Recognized As Safe) and possess excellent growth and fermentation characteristics. Lactic acid bacteria metabolism produces various functional substances, including gamma-aminobutyric acid (GABA), polyphenols and polyunsaturated fatty acids, short-chain fatty acids, bacteriocins, vitamins, and extracellular polysaccharides. These substances play an important role in enhancing food flavor, extending shelf life, increasing food viscosity, and improving health benefits.

[0004] The rich nutritional components of Chlorella proteoglycans make it an ideal substrate for lactic acid bacteria fermentation. Fermenting algae with lactic acid bacteria can enhance the flavor of algal foods, strengthen the nutritional properties of algae, and improve the bioavailability of nutrients. However, lactic acid fermentation, as an important direction for the deep processing of Chlorella proteoglycans, still has many shortcomings. On the one hand, there are very few reports on excellent lactic acid bacteria strains suitable for fermenting algae, and there is an urgent need to screen strains suitable for fermenting Chlorella proteoglycans; on the other hand, systematic research on the key process parameters and flavor of lactic acid bacteria fermentation of Chlorella proteoglycans is still insufficient. Currently, existing technologies have not effectively solved these problems, hindering the widespread application of Chlorella proteoglycans in fermented foods. Summary of the Invention

[0005] The primary objective of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide a strain of plant lactobacillus.

[0006] Another object of the present invention is to provide the application of the aforementioned *Lactobacillus plantarum* in fermented beverages containing *Chlorella proteoglycans*.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A strain of Lactiplantibacillus plantarum, named Lactiplantibacillus plantarum HN3, with accession number GDMCC No:64816, was deposited on July 26, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0009] The 16S rRNA sequence of *Lactobacillus plantarum* is shown in SEQ ID NO.1.

[0010] A method for culturing *Lactobacillus plantarum* includes the following steps: inoculating *Lactobacillus plantarum* into a culture medium and culturing it at 30–37°C.

[0011] The culture medium is at least one of MRS culture medium, MRS broth culture medium or Chlorella proteoglycans culture medium; wherein the Chlorella proteoglycans culture medium is prepared by the following method: 40g of Chlorella proteoglycans powder is mixed with 960g of water, then homogenized under high pressure at 850-1200 bar for 30-90s (preferably 850 bar for 90s), and then the broken algal solution is pasteurized to obtain Chlorella proteoglycans culture medium.

[0012] The pasteurization conditions are: sterilization at 65-90℃ for 10-30 minutes; preferably: sterilization at 65℃ for 30 minutes.

[0013] The culture time is 0 to 24 hours (excluding 0 hours); preferably 16 hours.

[0014] A microbial preparation containing the aforementioned Lactobacillus plantarum.

[0015] The microbial preparation contains ≥1×10⁻⁶ Lactobacillus plantarum. 8 CFU / mL (or ≥1×10⁻⁶) 8 CFU / g).

[0016] The method for preparing the microbial preparation includes the following steps: inoculating the above-mentioned *Lactobacillus plantarum* into a culture medium for cultivation to obtain the microbial preparation; or further collecting *Lactobacillus plantarum* cells from the culture medium, washing and freeze-drying them to obtain the microbial preparation.

[0017] The culture medium is Chlorella proteoglycans culture medium, which is prepared by the following method: 40g of Chlorella proteoglycans powder is mixed with 960g of water, and then homogenized under high pressure at 850-1200 bar for 30-90s (preferably 850 bar for 90s). The broken algal solution is then pasteurized to obtain Chlorella proteoglycans culture medium.

[0018] The pasteurization conditions are: sterilization at 65-90℃ for 10-30 minutes; preferably: sterilization at 65℃ for 30 minutes.

[0019] The culture time is 0 to 24 hours (excluding 0 hours); preferably 16 hours.

[0020] The application of the aforementioned *Lactobacillus plantarum* and / or microbial preparations in fermented beverages containing *Chlorella proteoglycans*.

[0021] A fermentation method for a Chlorella protein-rich beverage includes the following steps:

[0022] Chlorella pyrenoidosa powder, sucrose, and water are mixed evenly, then homogenized under high pressure at 850–1200 bar for 30–90 seconds, followed by pasteurization and cooling to obtain a homogenized Chlorella pyrenoidosa liquid. Then, the above-mentioned Lactobacillus plantarum or microbial preparation is inoculated into the Chlorella pyrenoidosa homogenized liquid and statically fermented at 30–37°C to obtain a Chlorella pyrenoidosa beverage.

[0023] The amount of Chlorella proteinensis powder added accounts for 2-10% of the mass of the fermentation system; preferably 4-10%; more preferably 6% of the mass of the fermentation system.

[0024] The amount of sucrose added accounts for 2-12% of the mass of the fermentation system; preferably 2-10% of the mass of the fermentation system; more preferably 6% of the mass of the fermentation system.

[0025] The preferred high-pressure homogenization conditions are: pressure 850 bar and homogenization time 90 s.

[0026] The pasteurization conditions are: temperature 65-90℃, time 10-30 min; preferably: temperature 90℃, time 10 min.

[0027] The inoculum size of *Lactobacillus plantarum* is 1-6% of the fermentation system volume (to make the initial concentration of *Lactobacillus plantarum* HN3 ≥ 1 × 10⁻⁶). 6 (CFU / mL); preferably 2% of the fermentation system volume.

[0028] The static fermentation time is 6 to 16 hours; preferably 6 to 12 hours; more preferably 10 hours.

[0029] The present invention has the following advantages and effects compared with the prior art:

[0030] 1. Addressing the current technical bottleneck of lacking a dedicated lactic acid bacteria strain for fermenting Chlorella vulgaris powder, this invention provides a strain of *Lactiplantibacillus plantarum* HN3 isolated from an outdoor *Chlorella vulgaris* cultivation pond in Chengmai County, Hainan Province. This strain exhibits good acid and bile salt tolerance, making it a safe strain for consumption. It can perform lactic acid fermentation using *Chlorella vulgaris* culture medium, demonstrating excellent fermentation performance, good flavor of the fermentation broth, and high sensory acceptance.

[0031] 2. This invention also provides a novel method for fermenting Chlorella proteinensis beverage using Bacillus plantarum HN3. The fermented Chlorella proteinensis beverage prepared by this method has a golden color, no layering, a harmonious and natural flavor, no fishy smell, and has the aroma brought by plantarum fermentation and the fragrance of Chlorella proteinensis itself. It has a delicate taste and a moderate sweet and sour ratio.

[0032] 3. The plant lactobacillus HN3 in this invention has excellent fermentation performance, acid resistance and bile salt tolerance. It can degrade unpleasant odor substances in the broken Chlorella pyrenoidosa powder liquid, produce new pleasant odor substances, significantly improve the flavor of the fermentation liquid, enhance the nutritional characteristics of Chlorella pyrenoidosa, improve the stability and sensory acceptance of algal fermentation beverages, and broaden the food application range of Chlorella pyrenoidosa. Attached Figure Description

[0033] Figure 1 This is a colony morphology diagram of Lactobacillus plantarum HN3.

[0034] Figure 2 This is the complete genome map of Lactobacillus plantarum HN3. Detailed Implementation

[0035] The present invention will be further described in detail below with reference to embodiments, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field. Test methods in the following embodiments that do not specify specific experimental conditions are generally performed under conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention are all commercially available.

[0036] The *Auxenochlorella pyrenoidosa* strain used in this invention is a chlorophyll synthesis-deficient mutant strain CX41, which has been disclosed in Chinese patent (patent number: ZL 2023 105723078, titled: A Chlorophyll Synthesis-Deficient Mutant Strain of *Auxenochlorella pyrenoidosa* and its Application), with accession number GDMCC No:63357. This strain contains little or no chlorophyll, is rich in xanthophyll, exhibits high growth and protein synthesis rates, and has no fishy odor. The CX41 strain freeze-dried powder was prepared using a heterotrophic fermentation method: strain activation, shake-flask transfer, fermenter culture, centrifugation, washing, freeze-drying, and pulverization to obtain the CX41 strain freeze-dried powder (the specific culture method is the same as that of *Auxenochlorella pyrenoidosa* in Example 1 of the patent application).

[0037] The MRS broth culture medium, MRS agar plate culture medium, and Columbia blood agar culture medium involved in the embodiments of the present invention can all be obtained through conventional commercial means.

[0038] Example 1: Isolation, purification, and identification of strains

[0039] 1.1 Sample Source:

[0040] One mL of algal culture was taken from the outdoor Chlorella proteoglycans culture pond (outdoor racetrack pond) of Hainan Aoji Bioengineering Co., Ltd. in Chengmai County, Hainan Province. The culture was enriched in MRS broth at 37°C for 24 h. One hundred μL of the enriched culture was then spread onto MRS agar plates supplemented with 1% (w / v) calcium carbonate and incubated upside down at 37°C for 48 h. Single colonies with large calcium dissolution zones, round and protruding colonies with smooth edges were selected and streaked onto MRS agar plates for purification. Three batches were cultured, and the purified strains were stored in 20% (v / v) glycerol at -80°C.

[0041] 1.2 Identification of Lactic Acid Bacteria Strains

[0042] The purified strains were subjected to Gram staining and catalase assays. Gram-positive and catalase-negative strains were selected for bacterial genomic DNA extraction. The obtained genomic DNA was amplified by PCR, and the PCR products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results were uploaded to the GeneBank database, and BLAST was used for homology comparison to determine the species of the isolated strains.

[0043] 1.3 Results Analysis: A total of 11 strains were identified, including 5 strains of Lactococcus lactis subsp. lactis, 2 strains of Pediococcus acidilactici, 2 strains of Pediococcus pentosaceus, and 2 strains of Lactiplantibacillus plantarum (see Table 1).

[0044] Example 2 Evaluation of the growth and fermentation performance of lactic acid bacteria strains

[0045] Using the 11 strains in Example 1 as the research object, the growth and fermentation characteristics of each strain in Chlorella proteoglycans culture medium were tested, and the viable count, pH, total acidity, odor and other indicators of the fermentation broth were evaluated to screen out lactic acid bacteria strains suitable for Chlorella proteoglycans CX41 fermentation.

[0046] 2.1 Chlorella liquid fermentation

[0047] Preparation of Chlorella proteoglycans culture medium: Weigh 40g of Chlorella proteoglycans lyophilized powder and mix with 960g of purified water, then homogenize under high pressure at 850 bar for 90s, and pasteurize the algal solution at 65℃ for 30min.

[0048] After activating the lactic acid bacteria strains in MRS broth at 37℃ for two generations, they were inoculated into MRS broth medium at a 1% (v / v) inoculum and cultured at 37℃ for 16 h. The cells were collected by centrifugation (10000×g, 10 min, 4℃), washed twice with sterile physiological saline, and then resuspended in sterile physiological saline to adjust the cell density to 1×10⁻⁶. 8 CFU / mL. The bacterial suspension was inoculated into the above-mentioned Chlorella proteoglycans culture medium at an inoculum size of 1% (v / v) and fermented for 24 hours.

[0049] 2.2 Characterization of the growth and fermentation performance of lactic acid bacteria

[0050] Samples were taken at 0h and 24h of fermentation for analysis. The indicators included viable lactic acid bacteria count, pH value, total acidity, and odor evaluation to characterize the growth and fermentation performance of each strain. Specifically, the viable bacteria count in the fermentation broth was determined using the plate count method according to the national standard (GB 4789.35-2016); the pH value of the fermentation broth was measured using a pH meter; and the total acidity content was determined according to the national standard (GB 12456-2021).

[0051] 2.3 Results Analysis: The 11 bacterial strains exhibited different growth and fermentation properties in the Chlorella proteoglycans culture medium. Among them, strain HN3 showed the best overall performance and high sensory acceptance. After 24 hours of fermentation in the Chlorella proteoglycans culture medium, the viable cell count was greater than 1×10⁻⁶. 8 The fermentation broth had a concentration of CFU / mL, a pH of 3.84, and a total acid content of 5.33 g / L. The broth exhibited a pleasant aroma with sour, fruity, fatty, and grassy notes, without any unpleasant odors, significantly superior to the fermentation flavor of other strains. Therefore, HN3 was selected as the strain for fermenting Chlorella CX41 (Table 1).

[0052] Table 1. Evaluation of viable cell count, pH, total acid content, and odor of 11 bacterial strains after 24 hours of fermentation in Chlorella vulgaris medium.

[0053]

[0054] HN3 colony morphology as follows Figure 1 As shown, its 16S rRNA sequence SEQ ID NO.1 is as follows:

[0055]

[0056] Based on the colony morphology and molecular identification results of strain HN3, it was named Lactiplantibacillus plantarum HN3 and deposited on July 26, 2024, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No:64816.

[0057] Example 3: Evaluation of the acid and bile salt tolerance of Lactobacillus plantarum strain HN3

[0058] 3.1 Acid resistance

[0059] After two generations of activation in MRS broth, strain HN3 was inoculated into MRS broth at a 1% (v / v) inoculum and cultured at 37°C for 16 h. The cells were collected by centrifugation (10000×g, 10 min, 4°C), washed twice with PBS buffer (pH 7.2), and then resuspended in PBS solution (final concentration 1×10⁻⁶). 9 The inoculum (CFU / mL) was then inoculated at a 1% (v / v) inoculum into MRS broth medium adjusted to pH 2.5 and 3.0, and incubated at 37°C for 3 hours. At 0 and 3 hours, 1 mL samples were taken for viable cell count, and the survival rate was calculated (survival rate = logarithm of viable cell count at a certain time after inoculation / logarithm of initial viable cell count × 100%). Three replicates were set up for the experiment.

[0060] 3.2 Bile salt tolerance

[0061] After two generations of activation in MRS broth, strain HN3 was inoculated into MRS broth at a 1% (v / v) inoculum and cultured at 37°C for 16 h. The cells were collected by centrifugation (10000×g, 10 min, 4°C), washed twice with PBS buffer (pH 7.2), and then resuspended in PBS solution (final concentration 1×10⁻⁶). 9 CFU / mL was inoculated at a 1% (v / v) inoculation rate into MRS broth containing 0.3% and 0.5% (w / v) bile salts, and incubated at 37°C for 4 hours. 1 mL samples were collected at 0 and 4 hours for viable cell count, and the survival rate was calculated (survival rate = logarithm of viable cell count at a certain time after inoculation / logarithm of initial viable cell count × 100%). The experiment was conducted in triplicate.

[0062] 3.3 Results Analysis: After culturing for 3 hours at pH 2.5 and pH 3.0, the survival rates of strain HN3 were 98.30% and 98.92%, respectively, indicating that the strain has high acid resistance. After culturing for 4 hours at 0.3% and 0.5% bile salt concentrations, the survival rates were 94.18% and 86.53%, respectively, indicating that strain HN3 has high bile salt tolerance.

[0063] Example 4: Safety evaluation of Lactobacillus plantarum strain HN3

[0064] 4.1 Hemolysis test

[0065] After activating the HN3 strain for two generations in MRS broth, it was inoculated into MRS broth at a 1% (v / v) inoculum and cultured at 37°C for 16 h. Fresh seed culture was then inoculated into Columbia blood agar medium using an inoculation loop and cultured at 37°C for 48 h. The presence of hemolytic zones was then observed to determine whether the tested strain possessed hemolytic activity. The experiment was conducted in triplicate.

[0066] 4.2 Detection of enzyme activity of harmful metabolites

[0067] Kit Method: After activating the HN3 strain for two generations in MRS broth, inoculate the fresh bacterial culture into MRS broth and incubate at 37°C for 18–24 h; then dilute the fresh bacterial culture with 0.85% sterile physiological saline to a concentration of 1 × 10⁻⁶. 8 CFU / mL, pipette 0.05–0.08 mL (approximately 1–2 drops) and add it to the kit for ornithine decarboxylase, arginine decarboxylase, and lysine decarboxylase for determination. The experiment was conducted in triplicate.

[0068] 4.3 Safety evaluation based on whole-genome sequencing

[0069] Total DNA was extracted from strain HN3 for whole-genome sequencing. The genomic nucleic acid sequence was compared with the drug resistance gene database CARD, the virulence gene database VFDB, and ResFinder 4.1. Referring to the "Technical Guidelines for Safety Inspection and Evaluation of Microbial Strains Used in Health Food Raw Materials (2020 Edition)," the sequence length coverage ≥60%, sequence matching degree (≥85%), and e-value (<10-5) were used as the judgment criteria to predict whether strain HN3 contained drug resistance genes and virulence genes.

[0070] 4.4 Results Analysis: *Lactobacillus plantarum* HN3 did not exhibit hemolytic activity, nor did it possess ornithine decarboxylase, arginine decarboxylase, or lysine decarboxylase activity. The complete genome of strain HN3 was obtained through sequencing and assembly. Figure 2By searching for potential pathogenic or virulence genes in the genome using pathogenic gene data from Resfinder, VFDB, and CARD, it was proven that the genome does not contain any virulence or pathogenic genes. In summary, both phenotypic and genotypic levels demonstrate that strain HN3 is a biosafe strain.

[0071] Example 5: Process optimization for preparing Chlorella protein-nucleated beverages by fermentation with Lactobacillus plantarum HN3

[0072] 5.1 Process flow for preparing Chlorella protein-core fermented beverage

[0073] Process flow: Weighing of algae powder → Ingredient mixing → Mixing → High-pressure homogenization (850 bar, 90 s) → Chlorella proteoglycans homogenate → Pasteurization (90℃, 10 min) → Inoculation → Fermentation → Bottling → Chlorella proteoglycans fermented beverage. Specific steps: Weigh Chlorella proteoglycans powder, sucrose, and water, mix, and homogenize at 850 bar for 90 s. After pasteurization (treatment at 90℃ for 10 min), the resulting Chlorella proteoglycans homogenate is cooled to room temperature. Lactobacillus plantarum HN3 is inoculated into the Chlorella proteoglycans homogenate, mixed, and then allowed to ferment statically at 37℃. Bottling yields the Chlorella proteoglycans fermented beverage.

[0074] 5.2 Single-factor experiments on fermentation process parameters

[0075] 5.2.1 Fermentation time

[0076] 6% (w / w) sucrose was added to a homogenate containing 6% (w / w) Chlorella peptiflora powder. After pasteurization, the homogenate was inoculated with 3% (v / v) Lactobacillus plantarum HN3 and fermented at 37℃ for 8, 10, 12, 14 and 16 h, respectively, and then sensory evaluation was carried out.

[0077] 5.2.2 Inoculation Dosage

[0078] 6% (w / w) sucrose was added to a homogenate containing 6% (w / w) Chlorella peptone powder. After pasteurization, 1%, 2%, 3%, 4%, and 5% (v / v) Lactobacillus plantarum HN3 were inoculated, and the mixture was fermented at 37°C for 12 h before sensory evaluation.

[0079] 5.2.3 Algae powder addition amount

[0080] 6% (w / w) sucrose was added to homogenates containing 2%, 4%, 6%, 8%, and 10% (w / w) Chlorella peptiflora powder, pasteurized, and then inoculated with 3% (v / v) Lactobacillus plantarum HN3. Sensory evaluation was then performed after fermentation at 37°C for 12 hours.

[0081] 5.2.4 Sucrose addition amount

[0082] 2%, 4%, 6%, 8%, and 10% (w / w) of sucrose were added to a homogenate containing 6% (w / w) Chlorella peptiflora powder. After pasteurization, the homogenate was inoculated with 3% (v / v) Lactobacillus plantarum HN3 and fermented at 37°C for 12 h before sensory evaluation.

[0083] 5.3 Response Surface Design Experiment

[0084] Following single-factor experiments on fermentation process parameters, a response surface methodology (RSM) was developed to optimize the process parameters based on four key factors: fermentation time, inoculum size, algae powder, and sucrose addition, as well as their interactions. Sensory evaluation was used as the response value (Y) to achieve better process results. The RSM factors and levels are shown in Table 2.

[0085] Table 2 Horizontal Design of Response Surface Experiment

[0086]

[0087] 5.4 Analysis and Testing: The sensory flavor of the fermented milk beverage was evaluated by referring to the RHB104-2020 Fermented Milk Scoring Standard of China's Dairy Industry Standard, as shown in Table 3.

[0088] Table 3 Sensory Flavor Evaluation Table of Chlorella Proteinase Fermented Beverage

[0089]

[0090] 5.5 Results Analysis: Through single-factor and response surface methodology experiments on fermentation process parameters, the optimal process conditions for the lactic acid fermentation beverage of Chlorella proteoglycans were determined to be: inoculum size of 2.0% (v / v), sucrose addition of 6.0% (w / w), fermentation time of 10 h, and algae powder addition of 6% (w / w). Verification showed that under these conditions, the product achieved a sensory score of 87.3 points, with a golden appearance resembling mango juice, uniform color, no layering, a harmonious and natural flavor, no fishy algae odor, and possessing the fermentation aroma of *Lactobacillus plantarum* HN3 and the natural fragrance of *Chlorella proteoglycans*, a balanced sweet and sour ratio, and a delicate texture (Table 4).

[0091] Table 4. Response surface methodology results for fermentation process parameters of *Microcystis proteoglycans*

[0092]

[0093]

[0094] Example 6: Determination of flavor components in fermented Chlorella protein-nucleated beverages

[0095] 6.1 Preparation of Chlorella proteoglycans lactic acid fermentation beverage: Weigh 60g of Chlorella proteoglycans powder and 60g of sucrose and mix with 880g of purified water, then perform high pressure homogenization (850bar, 90s), pasteurize at 90℃ for 10min, cool to room temperature, inoculate Lactobacillus plantarum HN3 into the Chlorella proteoglycans homogenate at an inoculation rate of 2% (v / v), ferment at 37℃ for 10h and then fill into bottles to prepare Chlorella proteoglycans fermentation beverage.

[0096] 6.2 Analysis and Testing:

[0097] 6.2.1 Sample Pretreatment Procedure: Samples taken at 0h and 10h were used for volatile organic compound (VOC) detection. 1mL of sample was cryogenically ground in liquid nitrogen and vortexed until homogeneous. Approximately 0.2g (0.2mL liquid) of each sample was weighed into a headspace vial, and 0.2g of sodium chloride (NaCl) powder and 20μL of 3-hexanone (10μg / mL) internal standard solution were added. Sample extraction was performed using an automated headspace solid-phase microextraction (HS-SPME) system for GC-MS analysis.

[0098] HS-SPME extraction conditions: Shaking at 60℃ for 5 min, inserting a 120μm DVB / CWR / PDMS extraction head into the sample headspace vial, headspace extraction for 15 min; followed by analyzing at 250℃ for 5 min and then performing gas chromatography-mass spectrometry (GC-MS) for separation and identification. The extraction head was aged at 250℃ for 5 min in a fiber conditioning station before sampling. Note: New extraction heads, before extraction, are aged in a fiber conditioning station using SPMEArrow for 2 h; their sensitivity can reach 10 times that of traditional SPME fiber heads.

[0099] 6.2.2 Gas chromatography-mass spectrometry acquisition conditions:

[0100] Chromatographic conditions: DB-5MS capillary column (30m×0.25mm×0.25μm, Agilent J&W Scientific, Folsom, CA, USA), carrier gas: high-purity helium (purity not less than 99.999%), constant flow rate: 1.2mL / min, injection port temperature: 250℃, splitless injection, solvent delay: 3.5min. Temperature program: 40℃ held for 3.5min, increased to 100℃ at 10℃ / min, then increased to 180℃ at 7℃ / min, and finally increased to 280℃ at 25℃ / min, held for 5min.

[0101] Mass spectrometry conditions: Electron impact ion source (EI), ion source temperature 230℃, quadrupole temperature 150℃, mass spectrometry interface temperature 280℃, electron energy 70eV, scan mode selected ion detection mode (SIM), qualitative and quantitative ion precise scan (GB23200.8-2016).

[0102] Metabolites were identified by matching retention time, fragmentation pattern, and mass / charge ratio (m / z) values ​​with standards in the metabolite database of Wuhan Metware Biotechnology Co., Ltd.

[0103] 6.2.3 Calculation of the relative content of volatile organic compounds (VOCs)

[0104] The abundance of volatile organic compounds (VOCs) is determined based on their peak area relative to the internal standard compound. The formula for calculating the relative VOC content is as follows:

[0105]

[0106] In the formula, Xi is the content of compound i in the sample to be tested (μg / mL);

[0107] Vs is the volume (μL) of internal standard added;

[0108] Cs is the concentration of the internal standard (μg / mL);

[0109] V is the volume (mL) of the sample to be tested;

[0110] Is is the peak area of ​​the internal standard;

[0111] Ii represents the peak area of ​​compound i in the sample to be tested.

[0112] 6.2.4 Analysis of main odor substances

[0113] The relative odor activity (ROAV) value was used to evaluate the contribution of each volatile flavor component to the overall aroma of the fermented samples, and the ROAV value of the component that contributed the most to the fermentation flavor was defined. stan =100, then the ROAV of other volatile components is less than 100, and is calculated according to the following formula.

[0114]

[0115] In the formula: C i Indicates the relative content (%) of volatile component i;

[0116] Ti represents the threshold (μg / mL) of volatile component i;

[0117] C stan This indicates the relative content (%) of the component that contributes the most to the overall flavor of the sample;

[0118] T stan This represents the sensory threshold (μg / mL) of the component that contributes the most to the overall flavor of the sample.

[0119] Components with ROAV ≥ 1 are key flavor compounds in the analyzed samples, while components with 0.1 ≤ ROAV < 1 have an important modifying effect on the overall flavor of the samples.

[0120] 6.3 Results Analysis

[0121] By comparing the odor substances in the Chlorella vulgaris liquid before and after fermentation, it can be concluded that:

[0122] Regarding the main odor substances, after fermentation with *Lactobacillus plantarum* HN3, the *Chlorella proteoglycans* liquid retained its original pleasant flavor (roasted, fruity, sweet, mushroom, waxy, creamy, and herbal aromas) and produced new pleasant flavors (such as fatty aromas). At the same time, it degraded some of the off-odors (irritating odors) in the algal liquid, mainly by degrading isopentenyl thiol (an irritating odor substance), and enhanced two fatty aroma substances and one herbal aroma substance.

[0123] Regarding the modification of odor compounds, *Lactobacillus plantarum* HN3 also had a positive effect on the flavor modification after fermentation, increasing the amount of odor-modifying compounds with floral (1-hexanol) and fruity (δ-dodecyl lactone) aromas, thus enriching the flavor of the fermentation broth. This indicates that *Lactobacillus plantarum* HN3 has a significant flavor-improving effect on the fermentation broth of *Chlorella protozoa*, reducing off-odor compounds, increasing pleasant flavor compounds, and resulting in higher sensory acceptance (Table 5).

[0124] Table 5. Comparison of odor substances in the algal broth before and after fermentation with Lactobacillus plantarum HN3.

[0125]

[0126] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A strain of *Lactobacillus plantarum* ( Lactiplantibacillus plantarum ), characterized in that: The Lactiplantibacillus plantarum is named as Lactiplantibacillus plantarum HN3, and the preservation number is GDMCC No: 64816, which has been preserved in the Guangdong Microbial Culture Collection Center, 59 Building, 5 Floor, Guangzhou Xianlie Middle Road 100 Courtyard on July 26, 2024.

2. A method of culturing the Lactobacillus plantarum of claim 1, characterized by, The specific steps are as follows: inoculate the Lactiplantibacillus plantarum into the culture medium and culture at 30-37 ℃.

3. The method of claim 2, wherein: The culture medium is at least one of MRS culture medium, MRS bouillon culture medium or Chlorella pyrenoidosa culture medium. Auxenochlorella pyrenoidosa The Chlorella pyrenoidosa culture medium is prepared by mixing 40 g of Chlorella pyrenoidosa powder with 960 g of water, then homogenizing at 850-1200 bar for 30-90 s, and then pasteurizing the broken algal liquid to obtain the Chlorella pyrenoidosa culture medium. The pasteurization condition is 65-90 ℃ for 10-30 min.

4. A microbial preparation, characterized by: The Lactiplantibacillus plantarum of claim 1.

5. The method for preparing the microbial preparation according to claim 4, characterized in that: The method comprises the following steps: inoculating the Lactiplantibacillus plantarum into the culture medium to obtain a microbial preparation; or inoculating the Lactiplantibacillus plantarum into the culture medium to culture, and then collecting the Lactiplantibacillus plantarum in the culture solution, and washing and freeze-drying to obtain the microbial preparation.

6. The Lactiplantibacillus plantarum of claim 1 and / or the microbial preparation of claim 4 are used in the fermentation of Chlorella pyrenoidosa beverage, characterized in that: The Chlorella pyrenoidosa is a chlorophyll synthesis-deficient mutant strain CX41 of Chlorella pyrenoidosa, and the preservation number is GDMCC No: 63357.

7. A method of fermentation of a Chlorella pyrenoidosa drink, characterized in that, The method comprises the following steps: The Chlorella pyrenoidosa powder, sucrose and water are uniformly mixed, then high-pressure homogenization is performed at 850-1200 bar for 30-90 s, and then pasteurization, cooling are performed to obtain a Chlorella pyrenoidosa homogenate; then the Lactiplantibacillus plantarum of claim 1 or the microbial preparation of claim 4 is inoculated into the Chlorella pyrenoidosa homogenate, and static fermentation is performed at 30-37 ℃ to obtain a Chlorella pyrenoidosa beverage. The Chlorella pyrenoidosa is a chlorophyll synthesis-deficient mutant strain CX41 of Chlorella pyrenoidosa, and the preservation number is GDMCC No: 63357.

8. The fermentation method of claim 7, wherein: The addition amount of the Chlorella pyrenoidosa powder accounts for 2-10% of the mass of the fermentation system; The addition amount of the sucrose accounts for 2-12% of the mass of the fermentation system; The inoculation amount of the Lactiplantibacillus plantarum accounts for 1-6% of the volume of the fermentation system; The static fermentation time is 6-16 h.

9. The fermentation method of claim 8, wherein: The addition amount of the Chlorella pyrenoidosa powder accounts for 4-10% of the mass of the fermentation system; The addition amount of the sucrose accounts for 2-10% of the mass of the fermentation system; The static fermentation time is 6-12 h.

10. The fermentation method of claim 8, wherein: The addition amount of the Chlorella pyrenoidosa powder accounts for 6% of the mass of the fermentation system; The addition amount of the sucrose accounts for 6% of the mass of the fermentation system; The inoculation amount of the Lactiplantibacillus plantarum accounts for 2% of the volume of the fermentation system.

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