Cymbidium ensifolium fermentation liquor, preparation method and application thereof

Abstract

This invention discloses a lemongrass fermentation broth, its preparation method, and its application. The lemongrass fermentation broth simultaneously meets the following conditions: it contains live Lactiplantibacillus microorganisms, and the total number of live bacteria is ≥1×10⁻⁶. 9 The fermented lemongrass broth contains cfu / mL and contains volatile active ingredients, including at least linalool and methyl heptenone. Compared with unfermented lemongrass raw materials, the content of methyl heptenone in the lemongrass fermentation broth has increased from undetectable to over 10%, and the relative content of linalool has increased to over 20%. This invention's lemongrass fermentation broth possesses both a high viable bacterial count and a specific spectrum of volatile active ingredients. Through microbial fermentation, it achieves the targeted transformation of volatile components in lemongrass raw materials, particularly significantly increasing the content of linalool, methyl heptenone, and various ester aroma components, thus possessing broad prospects for industrial application.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a lemongrass fermentation broth, its preparation method, and its application. Background Technology

[0002] Lemongrass, belonging to the genus *Cyperus* of the Poaceae family, is an important spice plant. Its main volatile component is citral, and current research on its antibacterial properties primarily focuses on lemongrass essential oil. Studies have found that lemongrass volatile oil has significant inhibitory effects on *Pseudomonas aeruginosa* and *Early Blight of Tomato*; it also exhibits significant antibacterial activity against four common clinical pathogens: *Escherichia coli*, *Staphylococcus aureus*, *Pseudomonas aeruginosa*, and *Pseudomonas violaceum*. Lemongrass also possesses good anti-inflammatory activity, effectively scavenging DPPH free radicals, ABTS free radicals, and linoleic acid, demonstrating excellent antioxidant effects. It is widely used in spice production, insect repellent development, and the cosmetics industry, and is also applied in aquaculture to promote growth and replace antibiotics.

[0003] Currently, lemongrass is mainly used in two ways: as raw material and as essential oil. The former has the disadvantage of taking up a lot of space due to its loose texture and high transportation costs, while the latter has the problem of low essential oil extraction volume, which cannot meet the needs of large-scale applications. Therefore, developing new ways to apply lemongrass is of great practical significance.

[0004] Fresh pressing and fermentation are both common techniques in the food industry. Freshly pressed juice retains nearly 70-80% of the effective active ingredients in lemongrass, while fermentation can extend the shelf life of fresh juice. However, due to lemongrass's antibacterial activity, it may be incompatible with some lactobacilli, affecting the fermentation effect. Consequently, the application of lemongrass fermentation liquid obtained through fermentation processes has not been effectively and widely implemented. Summary of the Invention

[0005] In view of the technical problems existing in the prior art, the present invention provides a lemongrass fermentation liquid, its preparation method and application, which can effectively solve the technical problems existing in the prior art.

[0006] The technical solution of this invention is: A lemongrass fermentation liquid, wherein the lemongrass fermentation liquid is obtained by fermenting lemongrass raw materials with microorganisms of the genus Lactiplantibacillus, and the lemongrass fermentation liquid simultaneously meets the following conditions: (1) Contains microorganisms in a viable state, and the total number of viable bacteria is ≥1×10 9 cfu / mL; (2) Contains volatile active ingredients, wherein the volatile active ingredients include at least linalool and methyl heptenone; (3) Compared with unfermented lemongrass raw materials, the content of methyl heptenone in the lemongrass fermentation liquid has increased from never being detected to more than 10%, and the relative content of linalool has increased to more than 20%.

[0007] The lemongrass raw material is silage lemongrass juice, water extract of lemongrass, alcohol extract of lemongrass, steam distillation extraction by-product, or whole lemongrass pulp.

[0008] A method for preparing lemongrass fermentation liquid includes the following steps: (1) Activate the microorganisms of the genus Lactiplantibacillus to prepare seed liquid; (2) Inoculate the seed liquid from step (1) into lemongrass raw material at an inoculation amount of 1%~10% (v / v). (3) Ferment at 25~37℃ for 24~72 hours to obtain lemongrass fermentation liquid rich in live bacteria and volatile active ingredients.

[0009] In step (1), the microorganism is *Lactiplantibacillus plantarum*, specifically *Lactiplantibacillus plantarum* XM-1, whose 16S rDNA sequence is shown in SEQ ID NO:1. *Lactiplantibacillus plantarum* XM-1 was deposited at the China General Microbiological Culture Collection Center on March 17, 2026, with accession number CGMCC No. 37919. In step (3), after fermentation, the total viable count in the lemongrass fermentation broth is ≥1×10⁻⁶. 9 The concentration of methylheptenone was increased to over 10% compared to unfermented lemongrass raw material, and the relative content of linalool was increased to over 20%.

[0010] A *Lactiplantibacillus plantarum* XM-1, the 16S rDNA sequence of which is shown in SEQ ID NO:1, was deposited on March 17, 2026, at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37919.

[0011] The above-mentioned lemongrass fermentation liquid, or lemongrass fermentation liquid prepared by the above method, or the above-mentioned Lactobacillus plantarum XM-1, are used in the preparation of food, beverages, condiments, daily chemical products, feed additives or air fresheners or as functional raw materials.

[0012] Advantages of this invention: 1) This invention provides a novel lemongrass fermentation broth, which is prepared by fermenting lemongrass raw materials with Lactobacillus microorganisms. It simultaneously possesses a high viable cell count and a specific spectrum of volatile active ingredients: the total viable cell count in the fermentation broth can be stably maintained at 1×10⁻⁶. 9 The fermented broth contains cfu / mL or higher, and compared to unfermented lemongrass, the content of methyl heptenone is significantly increased to over 10%, and the relative content of linalool is significantly increased to over 20%. This fermented broth possesses both probiotic activity and characteristic aroma components, and can be directly used as a functional raw material in multiple fields such as food, daily chemicals, and feed.

[0013] 2) This invention utilizes the selected microorganism *Lactobacillus plantarum* XM-1 for fermentation, significantly altering the composition and content of volatile components in lemongrass raw materials, resulting in unexpected technical effects: Key active ingredients are significantly increased: the relative content of linalool is increased to over 20%, more than 4.5 times higher than unfermented lemongrass; methyl heptenone (a precursor for the synthesis of important compounds such as citral) is increased from undetectable to over 10%; ester aroma components are significantly increased: the relative contents of nerol acetate and geraniol acetate are increased to over 4% and over 8% respectively, an increase of 8-10 times, giving the fermentation broth a completely new aroma spectrum; other beneficial components are synergistically optimized: the relative contents of volatile components such as allocirrhene and α-terpineol are also significantly increased.

[0014] 3) The *Lactobacillus plantarum* XM-1 used in this invention exhibits excellent growth performance in lemongrass raw materials with natural antibacterial activity: the viable cell count can reach 1×10⁻⁶ after 48 hours of fermentation. 9 The concentration of cfu / mL is above 1×10⁻⁶ after 96 hours of fermentation. 9 With a cfu / mL or higher, the acid production effect is significant, which can reduce the pH value of the fermentation broth to below 4.0 and effectively extend the product's shelf life.

[0015] 4) The lemongrass fermentation liquid preparation method provided by this invention is simple, mild, and highly operable. It only requires inoculating the lemongrass raw material with *Lactobacillus* and allowing it to ferment at 25-37°C for 24-72 hours. No complex equipment is required, making it easy to scale up production. This invention effectively solves the technical bottlenecks in the application of lemongrass raw materials: compared to directly using the loose and costly lemongrass, the fermentation liquid is easier to store and transport; compared to lemongrass essential oil, which has a low extraction rate and limited yield, the fermentation method can fully utilize the lemongrass raw material, significantly improving the comprehensive utilization rate of lemongrass resources. Attached Figure Description

[0016] Figure 1 This is a morphological diagram of Lactobacillus plantarum XM-1 in an embodiment of the present invention.

[0017] Figure 2 Phylogenetic tree of Lactobacillus plantarum XM-1 and other lactobacilli (Genbank accession number for Lactobacillus plantarum XM-1 is PX755648).

[0018] Figure 3 The growth of Lactobacillus plantarum XM-1 was simulated in gastric juice at different pH values.

[0019] Figure 4 The hemolytic activity of Lactobacillus plantarum strain XM-1.

[0020] Figure 5 This refers to the pH value during the lemongrass juice fermentation process.

[0021] Figure 6 Dynamic fitting curves of effective viable Lactobacillus plantarum XM-1 count in lemongrass juice fermentation broth at different fermentation times. Detailed Implementation

[0022] To facilitate understanding by those skilled in the art, the structure of the present invention will now be described in further detail with reference to the accompanying drawings: Example 1: Isolation and Identification of Lactobacillus plantarum Isolation: The lemongrass used in the test was provided by the Institute of Resources, Environment and Soil Fertilizer, Fujian Academy of Agricultural Sciences. Fresh lemongrass was harvested, dried, sliced, and crushed. After 30-40 days of silage, 8-10 silage lemongrass leaves were placed in 50 mL of MRS liquid medium and incubated at 37℃ for 48 hours. The incubated bacterial solution was then spread onto MRS solid medium and incubated at 37℃ for 48 hours. Single colonies with similar morphological characteristics were streaked on the three zones of the MRS solid medium and incubated at 37℃ for 48 hours. This process was repeated multiple times until a single colony was observed on the plate, which was named *Lactobacillus plantarum* XM-1.

[0023] 2. Morphological identification: Observe the morphology of single colonies. The main morphology of the strain is as follows: colonies are small, with a diameter of about 2-3 mm, raised, round, smooth, with regular, dense, and white edges. Specifically, as shown below... Figure 1 As shown.

[0024] 3. Molecular biological identification: Single colonies from the same plate were selected, and DNA was extracted for PCR amplification. The amplified fragment was sequenced using the Sanger sequencing method, and the sequencing results are shown in SEQ ID NO:1. The sequenced sequence was compared with the GenBank database. The results showed that this strain had 100% similarity to Lactobacillus plantarum in the database. A phylogenetic tree was constructed using MEGA 6.0 software, as shown below. Figure 2 As shown.

[0025] 4. Preservation: Lactobacillus plantarum XM-1 was deposited at the China General Microbiological Culture Collection Center on March 17, 2026, with the accession number CGMCC No. 37919.

[0026] Example 2: Acid resistance of Lactobacillus plantarum XM-1 The pH of MRS liquid culture medium containing artificial protein gastric juice was adjusted to 1.5, 2.5 and 3.5 respectively using 1 mol / L hydrochloric acid. Activated Lactobacillus plantarum XM-1 was inoculated into the culture medium at different pH values ​​at a rate of 1%. The medium was incubated at 37℃ for 3 hours and then removed. The effective viable count was determined using the dilution plating method. The effective viable count of Lactobacillus cfu / mL = (number of colonies on the plate × dilution factor) / sampling volume.

[0027] The growth of the strain in artificial simulated gastric juice, such as Figure 3 As shown, after culturing in simulated gastric fluid at pH 1.5 for 3 hours, the survival rate of *Lactobacillus plantarum* XM-1 was 1.3 × 10⁻⁶. 6 The cfu / mL concentration was only 0.30%; after culturing in simulated gastric fluid at pH 2.5 for 3 hours, the survival rate of *Lactobacillus plantarum* XM-1 was 8.8 × 10⁻⁶. 7 The cfu / mL concentration yielded a survival rate of 20.54%; after culturing in simulated gastric fluid at pH 3.5 for 3 hours, the survival rate of *Lactobacillus plantarum* XM-1 was 2.28 × 10⁻⁶. 8 With a concentration of cfu / mL, the survival rate can reach 52.95%, which shows that Lactobacillus plantarum XM-1 has strong acid resistance.

[0028] Example 3: Hemolytic activity of Lactobacillus plantarum XM-1 Activated *Lactobacillus plantarum* XM-1 was inoculated onto blood agar plates and incubated at 37°C for 48 hours. The presence of a hemolytic zone around the colony was then observed. The hemolytic activity of the isolated strain was determined by assessing the lysis of red blood cells in the culture medium surrounding the colony, specifically based on the different phenomena of γ-hemolysis (safe), α-hemolysis, and β-hemolysis. *Staphylococcus aureus* was used as a positive control, and water was used as a negative control.

[0029] The results of the hemolytic activity test of the strain are as follows: Figure 4 As shown, on blood agar plates, no obvious transparent or green areas were observed around the Lactobacillus plantarum XM-1 colonies and the surrounding water, indicating that the isolated Lactobacillus plantarum XM-1 was a safe strain that was non-hemolytic (γ-hemolytic) after 48 hours of incubation, while hemolytic Staphylococcus aureus exhibited significant β-hemolytic activity. Therefore, Lactobacillus plantarum XM-1 is a non-hemolytic (γ-hemolytic) strain, a prerequisite characteristic of probiotics.

[0030] Example 4: Study on the re-fermentation of lemongrass juice by Lactobacillus plantarum XM-1 1. Seed culture preparation: The strains obtained from the above screening were inoculated into MRS liquid medium, activated for 2 generations, and cultured until the effective viable count was >1×10⁻⁶. 8 cfu / mL.

[0031] 2. Fermentation Experiment: The selected strains were inoculated into lemongrass juice at volume ratios of 1%, 3%, and 5% (denoted as T1, T2, and T3) for fermentation. The fermentation temperature was set at 37℃, and the fermentation was allowed to proceed for 24, 48, and 72 hours. A control group (CK) without the added strains was also fermented under the same conditions. Each treatment was repeated three times. The lemongrass juice was obtained by physically pressing the juice from the ensilinged fresh lemongrass as the fermentation raw material.

[0032] 3. Result determination: (1) pH measurement of fermentation broth: The lemongrass juice was removed from the incubator in batches according to fermentation times of 24h, 48h, and 72h. The pH value of the fermentation broth was measured using a pH meter, and the results are as follows: Figure 5 As shown: by Figure 5 It can be seen that during fermentation from 0 to 96 hours, the pH value of lemongrass juice in the CK treatment was greater than 5.0; while after adding *Lactobacillus plantarum* XM-1, the pH value of the juice decreased to below 4.0 after 48 hours of fermentation, and then tended to stabilize. Among them, at 48 hours, 72 hours, and 96 hours of fermentation, the pH values ​​of lemongrass juice in treatments T1, T2, and T3 were between 3.85 and 3.92, all significantly lower than that in CK. This shows that the application of *Lactobacillus plantarum* XM-1 to lemongrass juice fermentation can produce acid normally and reduce the pH value of the juice.

[0033] (2) Growth of Lactobacillus plantarum XM-1 in lemongrass juice Table 1 shows the changes in the effective viable count of *Lactobacillus plantarum* XM-1 in lemongrass juice after the application of *Lactobacillus*. As shown in Table 1, at 48h, 72h, and 96h of fermentation, the effective viable count of *Lactobacillus plantarum* XM-1 in the CK treatment was 0, while the effective viable counts of the lemongrass juice fermentation broth in the T1, T2, and T3 treatments were all >1×10⁻⁶. 9 The cfu / mL concentration indicates that *Lactobacillus plantarum* XM-1 can normally produce and reproduce in lemongrass juice. The T3 treatment, fermented for 48 hours, showed the highest number of viable bacteria, significantly higher than the T1 treatment, but not significantly different from the T2 treatment. The number of viable bacteria in the T3 treatments at 72 and 96 hours of fermentation was lower than that at 48 hours, and the difference between the T3 treatments was not significant. The number of viable bacteria was highest at 48 hours of fermentation. From 48 to 96 hours, the number of viable bacteria at all three addition levels decreased logarithmically with increasing fermentation time. Figure 6 As shown, the effective viable count of the fermentation product with the addition of the three strains is still >1×10⁻⁶. 9 cfu / mL.

[0034] Table 1 Effective viable bacteria count of lemongrass juice fermentation broth (1×10⁻⁶) 9 cfu / mL)

[0035] Note: Different letters after the data in the same column indicate significant differences, with a significance level of <0.05, and the same applies below.

[0036] (3) GS-MS analysis of volatile substances in fermentation products of Lactobacillus plantarum XM-1 added to lemongrass juice (3-1) Extraction of volatile substances: Volatile substances in lemongrass juice were extracted using solid phase microextraction (SPME). Extraction method: The extraction head of a 65µm PDMS / DVB was soaked in acetone for 30 min and then aged at 250℃ for 30 min; 10 mL of lemongrass juice was placed in a sealed 20 mL headspace vial, and after the headspace vial was heated in a 65℃ water bath for 5 min, the aged SPME fiber head was inserted, and after adsorption for 20 min, it was removed and ready for analysis.

[0037] (3-2) Determination of volatile substances: The volatile components of lemongrass juice were determined by GC-MS. Chromatographic conditions: initial temperature 50℃, hold for 2 min, increase to 120℃ at 5℃ / min, hold for 15 min, increase to 180℃ at 5℃ / min, hold for 2 min, increase to 260℃ at 30℃ / min, hold for 2 min. Mass spectrometry conditions: EI source; full scan mode; solvent delay 6 min; EMV mode, relative value; mass scan range: 50.00~550.00 amu; MS ion source temperature 230℃, MS quadrupole temperature 150℃. A mixture of n-alkanes diluted with n-hexane was used as a control sample to calculate the retention index of volatile substances.

[0038] (3-3) Qualitative and quantitative analysis The volatile components in lemongrass juice were identified by searching the NIST spectral library and calculating the experimental retention index (KI) of each component using a linear temperature rise formula. The relative content of each aroma component was calculated using the peak area normalization method, expressed as the ratio of the peak area of ​​each component to the total peak area. All experimental data were processed using Excel software and statistically analyzed using SPSS 19.0 software. Duncan's new multiple range method was used for comparisons among multiple groups, and t-tests were used for comparisons of paired samples. Data are expressed as mean values; P < 0.05 was considered statistically significant, and P > 0.05 was considered not statistically significant.

[0039] Based on the above results, adding 1 mL of *Lactobacillus plantarum* XM-1 can achieve the expected fermentation effect. Therefore, the recommended amount of *Lactobacillus plantarum* XM-1 bacterial solution used in this application is 1 mL, which is the T1 treatment. GC-MS analysis was performed on lemongrass juice fermented for 48 h, 72 h, and 96 h under the T1 treatment. The results are shown in Tables 2 and 3. Table 2 shows that after 48 h of fermentation, the contents of alkenes, alcohols, and aldehydes in the T1 treatment significantly decreased, while the contents of ketones significantly increased. There was no significant difference in the contents of esters and other compounds. After 72 h and 96 h of fermentation, the contents of aldehydes significantly decreased, while the contents of esters and ketones significantly increased. There was no significant difference in the contents of other compounds. This indicates that the effect of *Lactobacillus plantarum* on volatile components may mainly occur after 48 h of fermentation, and then tend to stabilize.

[0040] Table 2. Relative content (%) of major volatile substances in lemongrass juice

[0041] Note: Based on the above results, only the fermentation product with an addition amount of 1 mL was analyzed by GC-MS in the production process, and the same applies below.

[0042] Table 3. Composition and content of volatile substances in lemongrass juice

[0043]

[0044] Note: ND indicates not detected.

[0045] Table 3 shows that, with retention times ranging from 9 to 37 min, 28 to 29 known volatile components were identified in both the CK and T1 treatments. After fermentation for 48, 72, and 96 h, the relative contents of known volatiles in the two treatments ranged from 89.06% to 98.13%. For the CK treatment, the main volatile substances, in descending order of content, were R-citronellol / geraniol, citral / myrcene, isogeraniol, and Z-ocimene, with relative contents ranging from 50.33% to 53.04%. For the T1 treatment, the main volatile substances, in descending order of content, were methylheptenone / linalool, R-citronellol / geraniol, geraniol acetate / caryophyllene, and Z-ocimene, with relative contents ranging from 59.68% to 69.90%.

[0046] It should be noted that this embodiment is implemented in the same way as the first embodiment in terms of principle and technical effect. For the sake of brevity, any parts not mentioned in this embodiment can be referred to the corresponding content in the first embodiment.

[0047] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A lemongrass fermentation liquid, characterized in that, The lemongrass fermentation broth is prepared by fermenting lemongrass raw materials using microorganisms of the genus *Lactiplantibacillus*, and the lemongrass fermentation broth simultaneously meets the following conditions: (1) Contains microorganisms in a viable state, and the total number of viable bacteria is ≥1×10 9 cfu / mL; (2) Contains volatile active ingredients, wherein the volatile active ingredients include at least linalool and methyl heptenone; (3) Compared with unfermented lemongrass raw materials, the content of methyl heptenone in the lemongrass fermentation liquid has increased from never being detected to more than 10%, and the relative content of linalool has increased to more than 20%.

2. The lemongrass fermentation liquid according to claim 1, characterized in that, The lemongrass raw material is silage lemongrass juice, water extract of lemongrass, alcohol extract of lemongrass, steam distillation extraction by-product, or whole lemongrass pulp.

3. A method for preparing the lemongrass fermentation liquid according to claim 1 or 2, characterized in that, Includes the following steps: (1) Activate the microorganisms of the genus Lactiplantibacillus to prepare seed liquid; (2) Inoculate the seed liquid from step (1) into lemongrass raw material at an inoculation amount of 1%~10% (v / v). (3) Ferment at 25~37℃ for 24~72 hours to obtain lemongrass fermentation liquid rich in live bacteria and volatile active ingredients.

4. The method according to claim 3, characterized in that, The microorganism mentioned in step (1) is Lactiplantibacillus plantarum.

5. The method according to claim 4, characterized in that, The microorganism is *Lactobacillus plantarum* XM-1, whose 16S rDNA sequence is shown in SEQ ID NO:

1. *Lactobacillus plantarum* XM-1 was deposited at the China General Microbiological Culture Collection Center on March 17, 2026, with accession number CGMCC No. 37919.

6. The method according to any one of claims 3-5, characterized in that, After fermentation in step (3), the total number of viable bacteria in the lemongrass fermentation liquid is ≥1×10⁻⁶. 9 The concentration of methylheptenone was increased to over 10% compared to unfermented lemongrass raw material, and the relative content of linalool was increased to over 20%.

7. A *Lactiplantibacillus plantarum* XM-1, characterized in that, The 16S rDNA sequence of *Lactobacillus plantarum* XM-1 is shown in SEQ ID NO:

1. It was deposited on March 17, 2026, at the China General Microbiological Culture Collection Center with accession number CGMCC No. 37919.

8. The use of lemongrass fermentation liquid according to claim 1 or 2, or lemongrass fermentation liquid prepared by any one of claims 3 to 6, or Lactobacillus plantarum XM-1 according to claim 7 in the preparation of food, beverages, condiments, daily chemical products, feed additives or air fresheners or as a functional ingredient.

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