Phytobacterium plantarum and application thereof in chlorella pyrenoidosa fermented beverage

Abstract

The invention discloses a plant lactobacillus and an application of the plant lactobacillus in a chlorella pyrenoidosa fermented beverage. According to the invention, a strain of phytobacterium plantarum is separated and screened from an outdoor chlorella pyrenoidosa culture pond, is named as phytobacterium plantarum HN3, and is preserved in Guangdong Microbial Culture Collection Center on the fifth floor, No. 59 building, No. 100 Courtyard, Xianlie Middle Road, Guangzhou City on July 26, 2024, and the preservation number is GDMCC No: 64816. The plant lactobacillus HN3 has good fermentation performance, acid tolerance and cholate tolerance, can degrade unpleasant odor substances of chlorella pyrenoidosa liquid to generate new pleasant odor substances, and can be used as a production strain of a chlorella pyrenoidosa fermented beverage.

Description

Technical Field

[0001] The invention belongs to the field of fermentation and food biotechnology, and particularly relates to a plant lactobacillus strain and application thereof in a beverage fermented by pyrenoidosa Chlorella. Background Art

[0002] Auxenochlorella pyrenoidosa is a single-cell green algae of the Chlorophyta and Chlorella genus. It is a freshwater spherical single-cell algae with both plant and microbial properties and is widely distributed. The protein content in Auxenochlorella pyrenoidosa can reach 55-65% of the cell dry weight, and contains 18 kinds of amino acids, of which essential amino acids account for more than 40% of the total amino acids, exceeding 20% ​​of the cell dry weight. The essential amino acid index (EAAI) is 1.35, which is higher than the recommended protein intake standard. Auxenochlorella pyrenoidosa is also rich in dietary fiber and polyunsaturated fatty acids (such as linolenic acid and linoleic acid). In addition, Auxenochlorella pyrenoidosa also contains many nutrients and trace elements necessary for the human body, including vitamin B, vitamin C, vitamin D, vitamin E, niacin, folic acid, pantothenic acid, lutein, calcium, iron, zinc, etc. Auxenochlorella pyrenoidosa also has many physiological functions, such as cell regeneration, free radical scavenging, anti-tumor, promotion of antigen-specific immunity, T cell proliferation and activation, etc.

[0003] Lactic acid bacteria are a class of Gram-positive, non-spore-forming bacteria that can ferment carbon sources and produce a variety of organic acids such as lactic acid. They are widely used in the food fermentation industry. Commonly used lactic acid bacteria in the food industry mainly include strains of Streptococcus and Lactobacillus, such as Streptococcus salivarius thermophilus, Lactobacillus acidophilus, Lactobacillus delbrueckii bulgaricus, Lactobacillus casei, Lactobacillus rhamnosus, Lactobacillus helveticus and Lactobacillus plantarum. These strains are generally recognized as safe (GRAS) and have excellent growth and fermentation characteristics. Lactic acid bacteria metabolism can produce a variety of functional substances, including γ-aminobutyric acid (GABA), polyphenols and polyunsaturated fatty acids, short-chain fatty acids, bacteriocins, vitamins, exopolysaccharides and other substances, which play an important role in improving food flavor, extending shelf life, increasing food viscosity and improving health benefits.

[0004] The rich nutrients of Chlorella pyrenoidosa make it an ideal substrate for lactic acid bacteria fermentation. Using lactic acid bacteria to ferment algae can enhance the flavor of algae foods, strengthen the nutritional properties of algae, and increase the bioavailability of nutrients. Lactic acid fermentation, as one of the important directions of deep processing of Chlorella pyrenoidosa, still has many shortcomings. On the one hand, there are very few reports on excellent lactic acid bacteria strains suitable for fermenting algae, and it is urgent to screen strains suitable for fermenting Chlorella pyrenoidosa; on the other hand, systematic research on the key process parameters and flavor of lactic acid bacteria fermentation of Chlorella pyrenoidosa is still insufficient. At present, the existing technology has not been able to effectively solve these problems, which hinders the widespread application of Chlorella pyrenoidosa in fermented foods. Summary of the invention

[0005] The primary purpose of the present invention is to overcome the shortcomings and deficiencies of the prior art and provide a strain of Lactobacillus plantarum.

[0006] Another object of the present invention is to provide the use of the Lactobacillus plantarum in a beverage fermented by Chlorella pyrenoidosa.

[0007] The purpose of the present invention is achieved through the following technical solutions:

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

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

[0010] A method for culturing the plant lactobacillus comprises the following specific steps: inoculating the plant lactobacillus into a culture medium and culturing the culture medium at 30-37°C.

[0011] The culture medium is at least one of MRS culture medium, MRS broth culture medium or pyrenoid chlorella culture medium; wherein the pyrenoid chlorella culture medium is prepared by the following method: 40g pyrenoid chlorella powder is mixed with 960g water, and then 850-1200bar high pressure homogenization is used for 30-90s (preferably 850bar high pressure homogenization for 90s), and then the crushed algae liquid is pasteurized to obtain the pyrenoid chlorella culture medium.

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

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

[0014] A microbial preparation containing the above-mentioned Lactobacillus plantarum.

[0015] In the microbial preparation, the number of Lactobacillus plantarum is ≥ 1×10 8 CFU / mL (or ≥1×10 8 CFU / g).

[0016] The preparation method of the microbial preparation comprises the following steps: inoculating the plant lactobacillus into a culture medium for culturing to obtain the microbial preparation; or further collecting the plant lactobacillus cells in the culture solution, washing and freeze-drying to obtain the microbial preparation.

[0017] The culture medium is a pyrenoid chlorella culture medium, which is prepared by the following method: 40g of pyrenoid chlorella powder is mixed with 960g of water, and then 850-1200bar high-pressure homogenization is used for 30-90s (preferably 850bar high-pressure homogenization for 90s), and then the crushed algae liquid is pasteurized to obtain the pyrenoid chlorella culture medium.

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

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

[0020] Application of the plant lactobacillus and / or microbial preparation in Chlorella pyrenoidosa fermented beverage.

[0021] A fermentation method for a pyrenoid chlorella beverage comprises the following steps:

[0022] The pyrenoid chlorella powder, sucrose and water are uniformly mixed, and then homogenized at 850-1200 bar for 30-90 seconds, and then pasteurized and cooled to obtain a pyrenoid chlorella homogenate liquid; then the plant lactobacillus or microbial preparation is inoculated into the pyrenoid chlorella homogenate liquid, and static fermentation is carried out at 30-37° C. to obtain a pyrenoid chlorella beverage.

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

[0024] The added amount of sucrose is 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 high pressure homogenization conditions are preferably: pressure 850 bar, homogenization time 90 s.

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

[0027] The inoculation amount of the plant lactobacillus accounts for 1-6% of the volume of the fermentation system (so that the initial bacterial concentration of plant lactobacillus HN3 is ≥1×10 6 CFU / mL); preferably 2% of the volume of the fermentation system.

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

[0029] Compared with the prior art, the present invention has the following advantages and effects:

[0030] 1. Aiming at the technical bottleneck of the lack of lactic acid bacteria strains for fermenting pyrenoid chlorella wall-breaking algae powder, the present invention provides a plant lactobacillus (Lactiplantibacillus plantarum) HN3 derived from an outdoor pyrenoid chlorella cultivation pond in Chengmai County, Hainan Province. The strain has good acid tolerance and bile salt tolerance, is a safe strain for consumption, can use pyrenoid chlorella culture medium for lactic acid fermentation, has excellent fermentation performance, good fermentation liquid flavor, and high sensory acceptance.

[0031] 2. The present invention also provides a new method for fermenting pyrenoid algae beverage with Lactobacillus plantarum HN3. The pyrenoid algae fermented beverage prepared by the method has a golden color, no stratification phenomenon, harmonious and natural flavor, no algae smell, and has the fragrance brought by lactobacillus plantarum fermentation and the fragrance of pyrenoid algae itself, delicate taste, and moderate sour and sweet ratio.

[0032] 3. The plant lactobacillus HN3 in the present invention has excellent fermentation performance, acid resistance and bile salt tolerance, can degrade the bad odor substances in the pyrenoid Chlorella algae powder crushed liquid, produce new pleasant odor substances, significantly improve the flavor of the fermentation liquid, enhance the nutritional characteristics of pyrenoid Chlorella, improve the stability and sensory acceptance of algae fermented drinks, and broaden the food application range of pyrenoid Chlorella. BRIEF DESCRIPTION OF THE DRAWINGS

[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 DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with examples, 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 the art. The test methods for which specific experimental conditions are not specified in the following examples are usually in accordance with conventional experimental conditions. Unless otherwise specified, the reagents and raw materials used in the present invention can be obtained commercially.

[0036] The pyrenoid chlorella algae used in the present invention is a pyrenoid chlorella (Auxenochlorellapyrenoidosa, referred to as Ap) chlorophyll synthesis defective mutant strain CX41, which has been disclosed in a Chinese patent (patent number: ZL 2023 105723078, entitled: A pyrenoid chlorella chlorophyll synthesis defective mutant strain and its application), and the deposit number is GDMCC No: 63357. The algae species does not contain or contains a trace amount of chlorophyll, is rich in lutein, has a high growth rate and protein synthesis rate, and has no algae smell. The CX41 algae strain freeze-dried powder is prepared by heterotrophic fermentation method: algae species activation, shake flask transfer, fermentation tank culture, centrifugation, washing, freeze drying, and crushing to obtain the CX41 algae strain freeze-dried powder (the specific culture method is the same as the pyrenoid chlorella culture in Example 1 of the patent application).

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

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

[0039] 1.1 Sample source:

[0040] 1 mL of algae liquid was taken from the outdoor protein nucleus Chlorella cultivation pond (outdoor runway pond) of Hainan Aoji Bioengineering Co., Ltd. in Chengmai County, Hainan Province, and enriched and cultured in MRS broth medium at 37°C for 24 hours. 100 μL of enriched culture liquid was applied to MRS agar plates added with 1% (w / v) calcium carbonate, and inverted and cultured at 37°C for 48 hours. Single colonies with larger diameter of calcium dissolution circle, round protrusions of colonies and smooth edges were picked, streaked on MRS agar plates for purification and culture for 3 batches, and the purified strains were stored at -80°C with 20% (v / v) glycerol.

[0041] 1.2 Identification of lactic acid bacteria strains

[0042] The purified strains were subjected to Gram staining and catalase tests. Gram-positive, catalase-negative strains were selected to extract bacterial genomic DNA, 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 the BLAST tool was used for homology comparison to determine the species of the isolated strains.

[0043] 1.3 Analysis of results: 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 Growth and fermentation performance evaluation of lactic acid strains

[0045] The 11 strains in Example 1 were used as research objects, the growth and fermentation characteristics of each strain in the culture medium of Chlorella pyrenoidosa were tested, the viable count, pH, total acid, odor and other indicators of the fermentation broth were evaluated, and the lactic acid bacteria strains suitable for Chlorella pyrenoidosa CX41 fermentation were screened out.

[0046] 2.1 Chlorella liquid fermentation

[0047] Preparation of Chlorella culture medium: Weigh 40 g of Chlorella freeze-dried powder and mix it with 960 g of purified water, then homogenize it under high pressure at 850 bar for 90 seconds, and pasteurize the algae liquid at 65°C for 30 minutes.

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

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

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

[0051] 2.3 Result analysis: The 11 strains showed different growth and fermentation performances in the pyrenoid algae culture medium, among which the HN3 strain had the best comprehensive performance and high sensory acceptance. In the pyrenoid algae culture medium, the number of viable bacteria was greater than 1×10 8 CFU / mL, the pH value of the fermentation liquid was 3.84, the total acid content was 5.33g / L, and the fermentation liquid had sour, fruity, fatty, and grassy aromas without any unpleasant odors, which was significantly better than the fermentation flavor of other strains. Therefore, HN3 was selected as the strain for fermenting Chlorella CX41 (Table 1).

[0052] Table 1 The viable bacterial count, pH, total acid content and odor evaluation of 11 strains after 24h fermentation in Chlorella pyrenoidosa culture medium

[0053]

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

[0055]

[0056] Based on the colony morphological characteristics and molecular identification results of the HN3 strain, it was named Lactiplantibacillus plantarum HN3. It was deposited in the Guangdong Provincial Microbiological Culture Collection Center (GDMCC) on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou on July 26, 2024, with the preservation number GDMCC No: 64816.

[0057] Example 3 Evaluation of acid and bile resistance of Lactobacillus plantarum HN3 strain

[0058] 3.1 Acid resistance

[0059] After HN3 strain was activated in MRS broth for two generations, it 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 of 1×10 9 CFU / mL), and then inoculated in MRS broth medium adjusted to pH 2.5 and 3.0 at 1% (v / v), incubated at 37°C for 3h, and 1mL samples were taken for viable count after 0 and 3h, and the survival rate was calculated (survival rate = logarithm of viable count at a certain time after inoculation / logarithm of initial viable count × 100%). The experiment was repeated three times.

[0060] 3.2 Bile salt tolerance

[0061] After HN3 strain was activated in MRS broth for two generations, it 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 of 1×10 9 CFU / mL), inoculated in MRS broth containing 0.3% and 0.5% (w / v) bile salts at a 1% (v / v) inoculation amount, incubated at 37°C for 4 h, and 1 mL of sample was taken after 0 and 4 hours for viable count, and the survival rate was calculated (survival rate = logarithm of viable count at a certain time after inoculation / logarithm of initial viable count × 100%). The experiment was repeated three times.

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

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

[0064] 4.1 Hemolysis experiment

[0065] After the HN3 strain was activated for 2 generations in MRS broth medium, it was inoculated into MRS broth medium at 1% (v / v) and cultured at 37°C for 16 hours. Fresh seed liquid was taken with an inoculation loop and punctured into Columbia blood agar medium. After culturing at 37°C for 48 hours, whether a hemolytic zone appeared was observed to determine whether the test strain had hemolytic activity. The experiment was set up with three replicates.

[0066] 4.2 Detection of enzyme activity of harmful metabolites

[0067] Kit method: After the HN3 strain was activated for 2 generations in MRS broth, the fresh bacterial solution was inoculated into MRS broth and cultured at 37°C for 18-24 hours; then the fresh bacterial solution was diluted to 1×10 8 CFU / mL, draw 0.05-0.08mL (about 1-2 drops) and add to ornithine decarboxylase, arginine decarboxylase, lysine decarboxylase kits for determination. The experiment was set up in triplicate.

[0068] 4.3 Safety evaluation based on whole genome sequencing

[0069] The total DNA of the HN3 strain was extracted for whole genome sequencing, and the genomic nucleic acid sequence was compared with the resistance gene database CARD, the virulence gene database VFDB and ResFinder4.1. Referring to the "Technical Guidelines for Safety Inspection and Evaluation of Bacteria Used as Health Food Ingredients (2020 Edition)", the sequence length coverage ≥60%, sequence matching (≥85%) and e-value (<10-5) were used as judgment criteria to predict whether the HN3 strain contains resistance genes and virulence genes.

[0070] 4.4 Result analysis: Lactobacillus plantarum HN3 has no hemolytic activity, and does not have ornithine decarboxylase, arginine decarboxylase, or lysine decarboxylase activity. The complete genome of the HN3 strain was obtained by sequencing and assembly ( Figure 2), and retrieved potential pathogenic genes or virulence genes in the genome through pathogenic gene data such as Resfinder, VFDB, and CARD, proving that its genome does not contain any virulence or pathogenic genes. In summary, the HN3 strain has been proven to be a biosafe strain at both the phenotypic and genotypic levels.

[0071] Example 5 Optimization of the process for preparing Chlorella pyrenoidosa beverage by fermentation with Lactobacillus plantarum HN3

[0072] 5.1 Process flow of preparing Chlorella pyrenoidosa fermented beverage

[0073] Process flow: weighing algae powder → batching → mixing → high-pressure homogenization (850bar, 90s) → pyrenoid chlorella homogenate → pasteurization (90℃, 10min) → inoculation → fermentation → filling → pyrenoid chlorella fermented beverage. Specific steps: weigh pyrenoid chlorella powder, sucrose and water, mix them, high-pressure homogenization at 850bar for 90s, pasteurize the pyrenoid chlorella homogenate obtained by homogenization (treatment at 90℃ for 10min) and then cool it to room temperature, inoculate Lactobacillus plantarum HN3 into the pyrenoid chlorella homogenate, mix them and ferment them at 37℃, fill them, and obtain pyrenoid chlorella fermented beverage.

[0074] 5.2 Single factor experiment of fermentation process parameters

[0075] 5.2.1 Fermentation time

[0076] 6% (w / w) sucrose was added to the homogenate of Chlorella pyrenoidosa powder at a concentration of 6% (w / w), and the mixture was pasteurized and inoculated with 3% (v / v) Lactobacillus plantarum HN3. The mixture was fermented at 37°C for 8, 10, 12, 14, and 16 h, and the sensory evaluation was performed.

[0077] 5.2.2 Inoculation volume

[0078] 6% (w / w) sucrose was added to the homogenate of Chlorella pyrenoidosa powder at a concentration of 6% (w / w). After pasteurization, 1%, 2%, 3%, 4%, and 5% (v / v) of Lactobacillus plantarum HN3 were inoculated respectively, and sensory evaluation was performed after fermentation at 37°C for 12 hours.

[0079] 5.2.3 Amount of algae powder added

[0080] 6% (w / w) sucrose was added to the homogenate of Chlorella pyrenoidosa powder at an addition amount of 2%, 4%, 6%, 8%, and 10% (w / w), respectively, and 3% (v / v) Lactobacillus plantarum HN3 was inoculated after pasteurization. The samples were fermented at 37°C for 12 hours and then the sensory evaluation was performed.

[0081] 5.2.4 Amount of sucrose added

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

[0083] 5.3 Response surface design experiment

[0084] After the single factor experiment of fermentation process parameters, the response surface design was carried out for the four key factors, namely fermentation time, inoculation amount, algae powder and sucrose addition, and their interactions, and the process parameters were optimized with sensory evaluation as the response value (Y) in order to achieve better process effects. The response surface experimental 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 Chlorella vulgaris fermented beverages was evaluated by referring to the Chinese dairy industry standard RHB104-2020 fermented milk scoring standard, see Table 3.

[0088] Table 3 Sensory flavor evaluation table of Chlorella pyrenoidosa fermented beverage

[0089]

[0090] 5.5 Result analysis: Through the single factor and response surface design experiments of fermentation process parameters, the optimal process conditions for protein core algae lactic acid fermented beverage were obtained as follows: inoculation amount was 2.0% (v / v), sucrose addition was 6.0% (w / w), fermentation time was 10h, and algae powder addition was 6% (w / w). It was verified that under this condition, the product sensory score was 87.3 points, the appearance was golden like mango juice, the color was uniform, there was no stratification phenomenon, the flavor was coordinated and natural, there was no algae smell, it had the fragrance of plant lactobacillus HN3 fermentation and the fragrance of protein core algae itself, the sweet and sour ratio was moderate, and the texture was delicate (Table 4).

[0091] Table 4 Response surface results of fermentation process parameters of pyrenoid algae

[0092]

[0093]

[0094] Example 6 Determination of flavor components of Chlorella pyrenoidosa fermented beverage

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

[0096] 6.2 Analysis and testing:

[0097] 6.2.1 Sample pretreatment process: Take samples at 0h and 10h for volatile organic compound (VOC) detection. Take 1mL sample for liquid nitrogen freeze grinding, vortex mixing, weigh about 0.2g (0.2mL liquid) of each sample into a headspace bottle, add 0.2g sodium chloride (NaC1) powder and 20μL 3-hexanone (10μg / mL) internal standard solution, extract the sample on the fully automatic headspace solid phase microextraction (HS-SPME) instrument for GC-MS analysis.

[0098] HS-SPME extraction conditions: shake at 60℃ for 5min, insert 120μm DVB / CWR / PDMS extraction head into sample headspace bottle, extract headspace for 15min; analyze at 250℃ for 5min, and then perform gas chromatography-mass spectrometry separation and identification. Before sampling, the extraction head is aged at 250℃ for 5min in the fiber conditioning station. Note: Before extraction, the new extraction head uses SPMEArrow and is aged in the fiber conditioning station for 2h. Its sensitivity can reach 10 times that of the traditional SPME fiber head.

[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 is high-purity helium (purity not less than 99.999%), constant flow rate 1.2mL / min, injection port temperature 250℃, non-split injection, solvent delay 3.5min. Program temperature rise: 40℃ for 3.5min, increase to 100℃ at 10℃ / min, then increase to 180℃ at 7℃ / min, and finally increase to 280℃ at 25℃ / min, and hold for 5min;

[0101] Mass spectrometry conditions: electron impact ion source (EI), ion source temperature 230°C, quadrupole temperature 150°C, mass spectrometry interface temperature 280°C, electron energy 70 eV, scanning mode selected ion detection mode (SIM), qualitative and quantitative ion precise scanning (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 relative content of volatile organic compounds (VOC)

[0104] The abundance of volatile organic compounds was determined based on their peak area relative to the internal standard compound. The relative content of VOC was calculated as follows:

[0105]

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

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

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

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

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

[0111] Ii is 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 value (ROAV) was used to evaluate the contribution of each volatile flavor component to the overall aroma of the fermented sample, and the component ROAV that contributed the most to the fermented flavor was defined. stan =100, then the ROAV of other volatile components is less than 100 and is calculated as follows.

[0114]

[0115] Where: C i represents the relative content of volatile component i (%);

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

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

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

[0119] The components with ROAV≥1 are the key flavor compounds of the analyzed samples, and the 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 of Chlorella pyrenoidosa liquid before and after fermentation, we can know that:

[0122] In terms of the main odor substances, after fermentation with Lactobacillus plantarum HN3, the protein-nucleated Chlorella liquid retained the original pleasant flavor (baked aroma, fruity aroma, sweetness, mushroom aroma, wax aroma, cream aroma, herbal aroma), and produced new pleasant flavors (such as fat aroma); at the same time, it degraded some of the odors (irritating smells) in the algae liquid, mainly degrading isopentenyl mercaptan (a irritating odor substance), and enhancing two fatty odor substances and one herbal pleasant odor substance.

[0123] In terms of modified odor substances, Lactobacillus plantarum HN3 also has a positive effect on the modified flavor after fermentation, increasing the modified odor substances with floral aroma (1-hexanol) and fruity aroma (δ-dodecalactone), enriching the flavor of the fermentation broth. This shows that Lactobacillus plantarum HN3 has a significant improvement effect on the flavor of Chlorella pyrenoidosa fermentation broth, reducing odor substances, increasing pleasant flavor substances, and having a higher sensory acceptance (Table 5).

[0124] Table 5 Comparison of odor substances before and after fermentation of algae liquid by Lactobacillus plantarum HN3

[0125]

[0126] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A strain of Lactobacillus plantarum, characterized in that: The name is Lactiplantibacillus plantarum HN3, the preservation number is GDMCC No: 64816, and it was deposited on July 26, 2024 at the Guangdong Provincial Microbiological Culture Collection Center on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. A method for culturing the plant lactobacillus according to claim 1, characterized in that: The specific steps are: inoculating Lactobacillus plantarum into the culture medium and culturing it at 30-37°C.

3. The method according to claim 2, characterized in that: The culture medium is at least one of an MRS culture medium, an MRS broth culture medium or a pyrenoid chlorella culture medium; wherein the pyrenoid chlorella culture medium is prepared by the following method: 40 g of pyrenoid chlorella powder is mixed with 960 g of water, and then 850 to 1200 bar high pressure homogenization is performed for 30 to 90 seconds, and then the crushed algae liquid is pasteurized to obtain a pyrenoid chlorella culture medium; The pasteurization conditions are: 65-90° C. sterilization for 10-30 minutes.

4. A microbial preparation, characterized in that: Contains the plant lactobacillus according to claim 1.

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

6. Use of the plant lactobacillus according to claim 1 and / or the microbial preparation according to claim 4 in fermentation of Chlorella pyrenoidosa beverage.

7. A fermentation method for a pyrenoid chlorella beverage, characterized in that: The steps include: The pyrenoid chlorella powder, sucrose and water are uniformly mixed, and then 850-1200 bar high pressure homogenization is adopted for 30-90 seconds, and then the mixture is pasteurized and cooled to obtain a pyrenoid chlorella homogenate liquid; then the plant lactobacillus described in claim 1 or the microbial preparation described in claim 4 is inoculated into the pyrenoid chlorella homogenate liquid, and static fermentation is carried out at 30-37° C. to obtain a pyrenoid chlorella beverage.

8. The fermentation method according to claim 7, characterized in that: The amount of the pyrenoid chlorella powder added is 2-10% of the mass of the fermentation system; The added amount of sucrose accounts for 2-12% of the mass of the fermentation system; The inoculation amount of the plant lactobacillus accounts for 1-6% of the volume of the fermentation system; The static fermentation time is 6 to 16 hours.

9. The fermentation method according to claim 8, characterized in that: The amount of the pyrenoid chlorella powder added is 4-10% of the mass of the fermentation system; The added amount of sucrose accounts for 2-10% of the mass of the fermentation system; The static fermentation time is 6 to 12 hours.

10. The fermentation method according to claim 8, characterized in that: The amount of the pyrenoid chlorella powder added is 6% of the fermentation system mass; The added amount of sucrose accounts for 6% of the mass of the fermentation system; The inoculation amount of the plant lactobacillus accounts for 2% of the volume of the fermentation system.

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