A high-efficiency extraction method of citronella essential oil

Abstract

The application provides a high-efficiency extraction method of citronella essential oil, relates to the technical field of plant essential oil extraction, and comprises the following steps: taking fresh citronella grass subjected to liquid nitrogen freezing treatment, crushing to obtain citronella grass fragments; using rope-shaped basket fungus YL1-MJ-5 bacterial liquid to perform mild fermentation on the citronella grass fragments for 8-10 h to obtain fermented materials; adding endoglucanase to the fermented materials to perform first-stage enzymolysis, and then adding exoglucanase and beta-glucosidase to perform second-stage enzymolysis to obtain enzymolysis materials; and performing co-water distillation on the enzymolysis materials to obtain citronella essential oil. Liquid nitrogen freezing causes water in cells to form ice crystals, significantly increasing the specific surface area of raw materials; rope-shaped basket fungus YL1-MJ-5 mild fermentation promotes tissue loosening, but does not completely destroy the cell wall, avoiding premature leakage of inclusions, and combining with stage-by-stage enzymolysis maximizes the synergistic efficiency of the three enzymes, while avoiding premature leakage of essential oil; and the application provides a new idea for high-efficiency extraction of citronella essential oil.

Description

Technical Field

[0001] This invention relates to the field of plant essential oil extraction technology, and in particular to a highly efficient extraction method for lemongrass essential oil. Background Technology

[0002] Lemongrass (Cymbopogon citratus) is a plant belonging to the genus Citronella in the family Poaceae. Its essential oil is rich in terpenoids such as citral and citronellol, possessing various biological activities including antibacterial, anti-inflammatory, and mosquito-repellent properties, and is widely used in food, daily chemical, and pharmaceutical industries. The main extraction methods for lemongrass essential oil include steam distillation, organic solvent extraction, and supercritical CO2 extraction. Among these, steam distillation has become the mainstream method for industrial production due to its simple equipment, safe operation, and low cost.

[0003] Lemongrass essential oil is primarily stored in the secretory cavities of plant leaves, encased by dense cell walls and vascular bundle sheaths. Traditional steam distillation relies solely on the permeation of high-temperature steam to release the essential oil, resulting in insufficient cell wall disruption and a significant amount of essential oil remaining in the plant tissue, leading to generally low yields (0.6%-1.5%). Existing technologies attempt to improve yields through fermentation using various microorganisms or enzymatic pretreatment. However, in practice, it has been found that microbial fermentation and enzymatic hydrolysis result in the complete release of internal components, causing premature leakage of the essential oil and its volatilization loss before subsequent distillation. This approach fails to protect and release the plant's original secondary metabolites, limiting the potential for yield improvement.

[0004] In view of this, the present invention is hereby proposed. Summary of the Invention

[0005] One of the objectives of this invention is to provide a highly efficient method for extracting lemongrass essential oil, so as to at least solve one of the technical problems existing in the prior art.

[0006] In order to achieve the above-mentioned objectives of the present invention, the following technical solution is adopted:

[0007] This invention provides a highly efficient method for extracting lemongrass essential oil, comprising the following steps:

[0008] A. Take fresh lemongrass that has been frozen with liquid nitrogen, crush it to obtain lemongrass powder;

[0009] B. Use the bacterial solution of *Bacillus ligustus* YL1-MJ-5 to lightly ferment lemongrass shreds for 8-10 hours to obtain fermented material;

[0010] C. Add endoglucanase to the fermentation material for the first stage of enzymatic hydrolysis, then add exoglucanase and β-glucosidase for the second stage of enzymatic hydrolysis to obtain the hydrolyzed material.

[0011] D. The enzymatically hydrolyzed material is subjected to co-distillation to obtain lemongrass essential oil.

[0012] Furthermore, the liquid nitrogen freezing treatment time is 30~60 min;

[0013] Before liquid nitrogen freezing, the length of fresh lemongrass is 1-2 cm;

[0014] After crushing, pass the lemongrass through a 40-60 mesh sieve to obtain lemongrass powder.

[0015] Furthermore, the OD of the bacterial solution 600 It is 1.7~2.1;

[0016] The inoculation amount of the bacterial solution is 5-10% of the amount of fresh lemongrass used.

[0017] Furthermore, the solid content in the lemongrass shred fermentation system is 45-50%;

[0018] The temperature for the light fermentation is 25~28℃;

[0019] The process following the light fermentation also includes a strain inactivation step;

[0020] The strain inactivation process involves inactivation at 65-70°C for 15-20 minutes.

[0021] Furthermore, the amount of endoglucanase added is 3~8 U / g of the dry weight of the fermentation material.

[0022] Furthermore, the amount of exoglucanase added is 3-5 U / g of the dry weight of the fermentation material; the amount of β-glucosidase added is 4-6 U / g of the dry weight of the fermentation material.

[0023] Furthermore, the temperatures for the first and second stages of enzymatic hydrolysis are independently 45-55℃.

[0024] The stirring speed for the first and second stages of enzymatic hydrolysis is 80-120 rpm, respectively.

[0025] The enzymatic hydrolysis time for the first stage and the second stage is 3-5 h, respectively.

[0026] Further, co-distillation involves transferring the enzymatically hydrolyzed material into a co-distillation apparatus, adding water to submerge the material, adding ethanol, adjusting the pH to 4-5 using citric acid, and then distilling.

[0027] Furthermore, the amount of ethanol added is 8-12% of the weight of fresh lemongrass;

[0028] After adding ethanol, 0.01~0.05% w / v of defoamer is also added, calculated based on the total volume of the system.

[0029] Furthermore, the total distillation time is 2 to 2.5 hours.

[0030] This invention provides a highly efficient extraction method for lemongrass essential oil. Liquid nitrogen freezing causes water in cells to form ice crystals, which, during mechanical crushing, cause the cell walls to become brittle due to the mechanical action of the ice crystals, significantly increasing the specific surface area of ​​the raw material. Mild fermentation with *Basilella fragilis* YL1-MJ-5 promotes tissue loosening without completely destroying the cell walls, preventing premature leakage of contents. Combined with staged enzymatic hydrolysis, the synergistic efficiency of the three enzymes is maximized, while also preventing premature leakage of the essential oil. This provides a new approach for the efficient extraction of lemongrass essential oil. Detailed Implementation

[0031] Unless otherwise defined herein, the scientific and technical terms used in conjunction with this invention shall have the meanings commonly understood by one of ordinary skill in the art. The meaning and scope of terms shall be clear; however, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or foreign definitions. In this application, unless otherwise stated, the use of "or" means "and / or". Furthermore, the use of the term "comprising" and other forms is non-limiting.

[0032] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0033] This invention provides a highly efficient method for extracting lemongrass essential oil, comprising the following steps:

[0034] A. Take fresh lemongrass that has been frozen with liquid nitrogen and crush it into lemongrass powder;

[0035] B. Use the bacterial solution of *Bacillus ligustus* YL1-MJ-5 to lightly ferment lemongrass shreds for 8-10 hours to obtain fermented material;

[0036] C. Add endoglucanase to the fermentation material for the first stage of enzymatic hydrolysis, and then add exoglucanase and β-glucosidase for the second stage of enzymatic hydrolysis to obtain the hydrolyzed material.

[0037] D. Citronella essential oil is obtained by co-distillation of the enzymatically hydrolyzed material.

[0038] Liquid nitrogen freezing causes water in cells to form ice crystals, which then break down the cell walls during mechanical crushing, significantly increasing the specific surface area of ​​the raw material. Mild fermentation with *Bambusa multiplex* YL1-MJ-5 promotes tissue loosening without completely destroying the cell walls, preventing premature leakage of contents. Combined with staged enzymatic hydrolysis, the synergistic efficiency of the three enzymes is maximized, while also preventing premature leakage of essential oils. This provides a new approach for the efficient extraction of lemongrass essential oil.

[0039] In some specific implementations, the liquid nitrogen freezing treatment time is 30-60 minutes; before the liquid nitrogen freezing treatment, the length of the fresh lemongrass is 1-2 cm; after crushing, it is passed through a 40-60 mesh sieve to obtain lemongrass fragments.

[0040] In some specific embodiments, the OD of the bacterial solution 600 The inoculum concentration is 1.7~2.1; the inoculation amount of the bacterial solution is 5~10% of the amount of fresh lemongrass used.

[0041] In some specific embodiments, the solid content in the lemongrass shred fermentation system is 45-50%; in some specific embodiments, the temperature of the light fermentation is 25-28°C.

[0042] In some specific embodiments, the mild fermentation is followed by a strain inactivation step; in some specific embodiments, the strain inactivation includes inactivation at 65-70°C for 15-20 minutes.

[0043] In some specific embodiments, the amount of endoglucanase added is 3-8 U / g of the dry weight of the fermentation material. In some specific embodiments, the amount of exoglucanase added is 3-5 U / g of the dry weight of the fermentation material; and the amount of β-glucosidase added is 4-6 U / g of the dry weight of the fermentation material.

[0044] In some specific embodiments, the temperatures of the first and second enzymatic hydrolysis stages are independently 45-55°C; in some specific embodiments, the stirring speeds of the first and second enzymatic hydrolysis stages are independently 80-120 rpm; and in some specific embodiments, the hydrolysis times of the first and second enzymatic hydrolysis stages are independently 3-5 h.

[0045] In some specific embodiments, co-distillation includes transferring the enzymatically hydrolyzed material to a co-distillation apparatus, adding water to submerge the material, adding ethanol, adjusting the pH to 4-5 using citric acid, and then distilling.

[0046] Ethanol is used as an azeotropic agent to lower the distillation temperature. Under acidic conditions, ethanol reacts with citral to form citral diethyl acetal. This acetal compound exhibits significantly higher chemical stability than free citral, fundamentally solving the problem of aldehyde condensation in high-temperature aqueous phases. The resulting essential oil is light in color, has a pure aroma, and is resistant to spoilage.

[0047] In some specific embodiments, the amount of ethanol added is 8-12% of the weight of fresh lemongrass; after adding ethanol, 0.01-0.05% w / v of defoamer is also added, calculated based on the total system volume. In some specific embodiments, the total distillation time is 2-2.5 h.

[0048] The present invention will be further illustrated below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or purchased directly from the market.

[0049] Talaromyces funiculosus YL1-MJ-5: from China Industrial Microbial Culture Collection Center, accession number: CICC 41690, abbreviated as: Talaromyces funiculosus YL1-MJ-5.

[0050] Talaromyces funiculosus YL1-MJ-1: from China Industrial Microbial Culture Collection Center, accession number: CICC 41691, abbreviated as: Talaromyces funiculosus YL1-MJ-1.

[0051] Talaromyces funiculosus BJ9.1: from China Industrial Microbial Culture Collection Center, accession number: CICC 41743, abbreviated as: Talaromyces funiculosus BJ9.1.

[0052] Activation medium for the strain: 30g sucrose, 3g NaNO3, 0.5g MgSO4·7H4O, 0.5g KCl, 0.01g FeSO4·4H4O, 1g K4HPO4, 15g agar, add water to 1L, pH 6.0~6.5.

[0053] Preparation of bacterial suspension: Inoculate the bacterial strain onto slant agar medium and incubate at 28°C for 5-7 days. After the mycelium has grown sufficiently and produced spores, wash the spores off the slant with sterile deionized water, transfer the suspension to a sterile Erlenmeyer flask, and adjust the OD of the spore suspension. 600 Bacterial solution is obtained by mixing at 1.7~2.1.

[0054] Example 1

[0055] A method for extracting lemongrass essential oil, comprising the following steps:

[0056] 1. Physical pretreatment of raw materials

[0057] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2 cm lengths, place it in a sieve basket and immerse it entirely in liquid nitrogen to freeze for 50 minutes. After removing it, crush it and pass it through a 60-mesh sieve.

[0058] 2. Mild fermentation by the strain

[0059] Inoculate crushed lemongrass with a bacterial suspension of YL1-MJ-5 (OD) 600The inoculum was 8% (v / w, calculated based on the amount of fresh lemongrass used before fermentation), with a solid content of 50%. Fermentation was carried out at 25°C for 9 hours, followed by raising the temperature to 70°C and holding for 18 minutes.

[0060] 3. Enzymatic hydrolysis

[0061] Add deionized water to adjust the solid content of the fermented material to 30%, and then proceed with enzymatic hydrolysis according to the following steps:

[0062] First stage: Add endoglucanase (5 U / g dry weight of fermented material), stir at 50℃ and 100 rpm for 4 hours for enzymatic hydrolysis;

[0063] Second stage: Add exoglucanase (5 U / g dry weight of fermented material) and β-glucosidase (12 U / g dry weight of fermented material), and continue enzymatic hydrolysis at 50℃ and 100 rpm for 4 h.

[0064] Dry weight determination: Take 10g of fermented material, dry it in an oven at 105℃ until constant weight, weigh it, and calculate the solid content (dry matter mass / total material mass × 100%). This determination is required for each batch of material to calculate enzyme dosage.

[0065] 4. Distillation

[0066] After enzymatic hydrolysis, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (10% of the weight of fresh lemongrass) and 0.02% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (100 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 4℃ for 50 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0067] Example 2

[0068] A method for extracting lemongrass essential oil, comprising the following steps:

[0069] 1. Physical pretreatment of raw materials

[0070] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2cm lengths, place it in a sieve basket and immerse it entirely in liquid nitrogen to freeze for 60 minutes. After removing it, pulverize it at low temperature and pass it through a 40-mesh sieve.

[0071] 2. Mild fermentation by the strain

[0072] Inoculate crushed lemongrass with a bacterial suspension of YL1-MJ-5 (OD) 600 2.1), calculated by weight of fresh lemongrass, with an inoculation amount of 5% (v / w, calculated based on the amount of fresh lemongrass used before fermentation), and the solid content adjusted to 45%, fermented at 28℃ for 8 h, and then heated to 65℃ and held for 20 min.

[0073] 3. Enzymatic hydrolysis

[0074] Add deionized water to adjust the solid content of the fermented material to 35%, and then proceed with enzymatic hydrolysis according to the following steps:

[0075] First stage: Add endoglucanase (8 U / g dry weight of fermented material), stir at 50℃ and 120 rpm for 3 h for enzymatic hydrolysis;

[0076] Second stage: Add exoglucanase (6 U / g dry weight of fermented material) and β-glucosidase (15 U / g dry weight of fermented material), and continue enzymatic hydrolysis at 120 rpm for 3 h at 50℃.

[0077] Dry weight determination: Take 10g of fermented material, dry it in an oven at 105℃ until constant weight, weigh it, and calculate the solid content (dry matter mass / total material mass × 100%). This determination is required for each batch of material to calculate enzyme dosage.

[0078] 4. Distillation

[0079] After enzymatic hydrolysis, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (8% of the weight of fresh lemongrass) and 0.01% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (120 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 10℃ for 30 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0080] Example 3

[0081] A method for extracting lemongrass essential oil, comprising the following steps:

[0082] 1. Physical pretreatment of raw materials

[0083] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2cm lengths, place it in a sieve basket, and immerse it entirely in liquid nitrogen to freeze for 30 minutes. After removing it, pulverize it at low temperature and pass it through a 40-mesh sieve.

[0084] 2. Mild fermentation by the strain

[0085] Inoculate crushed lemongrass with a bacterial suspension of YL1-MJ-5 (OD) 600 The inoculum amount was 10% (v / w, calculated based on the amount of fresh lemongrass used before fermentation), and the solid content was adjusted to 50%. Fermentation was carried out at 25℃ for 10 h, followed by raising the temperature to 70℃ and holding for 15 min.

[0086] 3. Enzymatic hydrolysis

[0087] Add deionized water to adjust the solid content of the fermented material to 25%, and then proceed with enzymatic hydrolysis according to the following steps:

[0088] First stage: Add endoglucanase (3 U / g dry weight of fermented material), stir at 50℃ and 80 rpm for 5 hours for enzymatic hydrolysis;

[0089] Second stage: Add exoglucanase (4 U / g dry weight of fermented material) and β-glucosidase (8 U / g dry weight of fermented material), and continue enzymatic hydrolysis at 50℃ and 80 rpm for 5 h.

[0090] Dry weight determination: Take 10g of fermented material, dry it in an oven at 105℃ until constant weight, weigh it, and calculate the solid content (dry matter mass / total material mass × 100%). This determination is required for each batch of material to calculate enzyme dosage.

[0091] 4. Distillation

[0092] After enzymatic hydrolysis, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (12% of the weight of fresh lemongrass) and 0.05% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (80 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 4℃ for 60 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0093] Comparative Example 1

[0094] The difference from Example 1 is that *Bacillus ligustus* YL1-MJ-1 is used instead of *Bacillus ligustus* YL1-MJ-5. The other steps are the same as in Example 1.

[0095] Comparative Example 2

[0096] The difference from Example 1 is that *Bacillus ligustus* BJ9.1 is used instead of *Bacillus ligustus* YL1-MJ-5. The other steps are the same as in Example 1.

[0097] Comparative Example 3

[0098] The difference from Example 1 is that step 2, the mild fermentation of the strain, is not performed:

[0099] 1. Physical pretreatment of raw materials

[0100] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2 cm lengths, place it in a sieve basket and immerse it entirely in liquid nitrogen to freeze for 50 minutes. After removing it, pulverize it at low temperature and pass it through a 60-mesh sieve.

[0101] 2. Enzymatic hydrolysis

[0102] Add deionized water to the crushed lemongrass to adjust the solid content of the fermented material to 30%, and then carry out enzymatic hydrolysis according to the following steps:

[0103] First stage: Add endoglucanase (5 U / g dry weight of fermented material), stir at 50℃ and 100 rpm for 4 hours for enzymatic hydrolysis;

[0104] Second stage: Add exoglucanase (5 U / g dry weight of fermented material) and β-glucosidase (12 U / g dry weight of fermented material), and continue enzymatic hydrolysis at 50℃ and 100 rpm for 4 h.

[0105] Dry weight determination: Take 10g of fermented material, dry it in an oven at 105℃ until constant weight, weigh it, and calculate the solid content (dry matter mass / total material mass × 100%). This determination is required for each batch of material to calculate enzyme dosage.

[0106] 3. Distillation

[0107] After enzymatic hydrolysis, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (10% of the weight of fresh lemongrass) and 0.02% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (100 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 4℃ for 50 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0108] Comparative Example 4

[0109] The difference from Example 1 is that step 3, enzymatic hydrolysis, is not performed:

[0110] 1. Physical pretreatment of raw materials

[0111] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2 cm lengths, place it in a sieve basket and immerse it entirely in liquid nitrogen to freeze for 50 minutes. After removing it, pulverize it at low temperature and pass it through a 60-mesh sieve.

[0112] 2. Mild fermentation by the strain

[0113] Inoculate crushed lemongrass with a bacterial suspension of YL1-MJ-5 (pre-activated to the logarithmic growth phase, OD adjusted) 600 The inoculum content is 1.7~2.1%, calculated based on the weight of fresh lemongrass. The inoculum amount is 8% (v / w, calculated based on the amount of fresh lemongrass used before fermentation). The solid content is adjusted to 50%. Fermentation is carried out at 25℃ for 9 h, and then the temperature is raised to 70℃ and held for 18 min.

[0114] 3. Distillation

[0115] After fermentation, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (10% of the weight of fresh lemongrass) and 0.02% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (100 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 4℃ for 50 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0116] Comparative Example 5

[0117] The difference from Example 1 is that, 3, enzymatic hydrolysis:

[0118] Deionized water was added to adjust the solid content of the fermented material to 35%. At the same time, endoglucanase (EG, 8 U / g dry weight of fermented material), exoglucanase (CBH, 6 U / g dry weight of fermented material) and β-glucosidase (BG, 15 U / g dry weight of fermented material) were added, and the mixture was stirred at 50℃ and 120 rpm for 6 h for enzymatic hydrolysis.

[0119] Comparative Example 6

[0120] The difference from Example 1 is that enzymatic hydrolysis is performed before fermentation:

[0121] 1. Physical pretreatment of raw materials

[0122] Clean the fresh lemongrass thoroughly, remove impurities, cut it into 1-2 cm lengths, place it in a sieve basket and immerse it entirely in liquid nitrogen to freeze for 50 minutes. After removing it, pulverize it at low temperature and pass it through a 60-mesh sieve.

[0123] 2. Enzymatic hydrolysis

[0124] Add deionized water to the crushed lemongrass to adjust the solid content of the fermented material to 30%, and then carry out enzymatic hydrolysis according to the following steps:

[0125] First stage: Add endoglucanase (5 U / g dry weight of fermented material), stir at 50℃ and 100 rpm for 4 hours for enzymatic hydrolysis;

[0126] Second stage: Add exoglucanase (5 U / g dry weight of fermented material) and β-glucosidase (BG, 12 U / g dry weight of fermented material), and continue enzymatic hydrolysis at 50℃ and 100 rpm for 4 h.

[0127] Dry weight determination: Take 10g of fermented material, dry it in an oven at 105℃ until constant weight, weigh it, and calculate the solid content (dry matter mass / total material mass × 100%). This determination is required for each batch of material to calculate enzyme dosage.

[0128] 3. Mild fermentation by the strain

[0129] Inoculate the enzymatically hydrolyzed material with a suspension of *Basilella fragilis* YL1-MJ-5 (pre-activated to the logarithmic growth phase, OD adjusted). 600 The inoculum content is 1.7~2.1%, calculated based on the weight of fresh lemongrass, with an inoculum amount of 8% (v / w, calculated based on the amount of fresh lemongrass used before fermentation). Fermentation is carried out at 25℃ for 9 h, followed by raising the temperature to 70℃ and holding for 18 min.

[0130] 4. Distillation

[0131] After enzymatic hydrolysis, the material was transferred to a co-water still. Sufficient deionized water was added to completely submerge the material. Ethanol (10% of the weight of fresh lemongrass) and 0.02% polydimethylsiloxane emulsion were added, followed by citric acid to adjust the pH to 4-5. Stirring was started (100 rpm), and the mixture was heated to a gentle boil (98-100℃). Timing was then initiated. The gentle boil was maintained, and the total distillation time was controlled at 2-2.5 hours. Distillation was stopped when a significant reduction in oil droplets was observed in the distillate. The generated vapor was condensed in a tubular condenser (cooling medium: 0-5℃ ice water). The condensate flowed into an oil-water separator and was allowed to stand at 4℃ for 50 minutes to separate into layers. The upper layer was lemongrass essential oil, which was then passed through anhydrous sodium sulfate to remove moisture, yielding lemongrass essential oil.

[0132] Comparative Example 7

[0133] Unlike Example 1, the fermentation time in step 2 is extended to 12 hours.

[0134] Comparative Example 8

[0135] Unlike Example 1, the fermentation time in step 2 is shortened to 6 hours.

[0136] Comparative Example 9

[0137] Unlike Example 1, step 1 does not use liquid nitrogen freezing; the material is directly pulverized and passed through a 60-mesh sieve.

[0138] Comparative Example 10

[0139] Citronella essential oil is extracted using the co-distillation method, specifically following these steps:

[0140] 1. Take fresh lemongrass and cut it into sections 1-2 cm in length.

[0141] 2. Place the mixture in a co-aqueous still, add deionized water (material-to-liquid ratio 1:20), and add NaCl with a final concentration of 9% (w / v). Heat to a gentle boil (98~100℃), start timing, maintain a gentle boil, and control the total distillation time to 2~2.5 h. Stop when a significant reduction in oil droplets is observed in the distillate. The generated steam is condensed through a tubular condenser (cooling medium is 0~5℃ ice water). The condensate flows into an oil-water separator, and is allowed to stand at 4℃ for 50 min to separate into layers. The upper layer is lemongrass essential oil, which is then passed through anhydrous sodium sulfate to remove water, yielding lemongrass essential oil.

[0142] All the above examples and comparative examples used the same batch of fresh lemongrass. 100g of fresh lemongrass was placed in an oven at 105℃ and dried to constant weight. The mass of the dried lemongrass was 33.18g. The dry weight was calculated as 33.18% of the weight of fresh lemongrass.

[0143] The yield of all essential oils is calculated using the following formula:

[0144] Essential oil yield (%) = lemongrass essential oil mass (g) / lemongrass dry weight (g) × 100%.

[0145] The results are shown in Table 1.

[0146] Table 1

[0147] Data shows that, compared with the conventional co-distillation method in Comparative Example 10, Examples 1-3 increased the yield of lemongrass essential oil by at least 2 times, significantly improving the extraction efficiency of lemongrass essential oil. Compared to Example 1, Comparative Examples 1 and 2 used other strains of the same genus. Due to differences in the metabolites of the strains, the extraction efficiency was significantly reduced. Comparative Example 3 had no fermentation step, Comparative Example 4 had no enzymatic hydrolysis step, and Comparative Example 6 had enzymatic hydrolysis followed by fermentation. Compared with Comparative Example 10, the yield was improved, but much lower than that of Example 1. This indicates that in order to exert a synergistic promoting effect on improving the yield of lemongrass essential oil, the fermentation and enzymatic hydrolysis steps are indispensable and the order of operation cannot be replaced. Comparative Example 5 had three enzymes added simultaneously, and its yield was also higher than that of Comparative Example 10, but much lower than that of Example 1. This indicates that the essential oil yield can be maximized by using a staged enzymatic hydrolysis step. The fermentation time of Comparative Examples 7 and 8 was too long or too short, both of which led to a decrease in the yield of essential oil. This indicates that maintaining a light fermentation and ensuring that the contained essential oil substances are not lost prematurely plays a key role. In Comparative Example 9, the absence of liquid nitrogen freezing also reduced the yield, but had a positive effect on the release of essential oil substances.

[0148] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A highly efficient extraction method for lemongrass essential oil, characterized in that, Includes the following steps: A. Take fresh lemongrass that has been frozen with liquid nitrogen, crush it to obtain lemongrass powder; B. Use the bacterial solution of *Bacillus ligustus* YL1-MJ-5 to lightly ferment lemongrass shreds for 8-10 hours to obtain fermented material; C. Add endoglucanase to the fermentation material for the first stage of enzymatic hydrolysis, then add exoglucanase and β-glucosidase for the second stage of enzymatic hydrolysis to obtain the hydrolyzed material. D. The enzymatically hydrolyzed material is subjected to co-distillation to obtain lemongrass essential oil.

2. The efficient extraction method according to claim 1, characterized in that, The liquid nitrogen freezing treatment time is 30~60 minutes; Before liquid nitrogen freezing, the length of fresh lemongrass is 1-2 cm; After crushing, pass the lemongrass through a 40-60 mesh sieve to obtain lemongrass powder.

3. The efficient extraction method according to claim 1, characterized in that, The OD of the bacterial solution 600 It is 1.7~2.1; The inoculation amount of the bacterial solution is 5-10% of the amount of fresh lemongrass used.

4. The efficient extraction method according to claim 3, characterized in that, The solid content in the lemongrass shred fermentation system is 45-50%. The temperature for the light fermentation is 25~28℃; The process following the light fermentation also includes a strain inactivation step; The strain inactivation process involves inactivation at 65-70°C for 15-20 minutes.

5. The efficient extraction method according to claim 1, characterized in that, The amount of endoglucanase added is 3~8 U / g of the dry weight of the fermentation material.

6. The efficient extraction method according to claim 1, characterized in that, The amount of exoglucanase added is 3-5 U / g of the dry weight of the fermentation material; the amount of β-glucosidase added is 4-6 U / g of the dry weight of the fermentation material.

7. The efficient extraction method according to claim 1, characterized in that, The temperatures for the first and second stages of enzymatic hydrolysis are independently 45~55℃; The stirring speed for the first and second stages of enzymatic hydrolysis is 80-120 rpm, respectively. The enzymatic hydrolysis time for the first stage and the second stage is 3-5 h, respectively.

8. The efficient extraction method according to claim 1, characterized in that, Co-distillation involves transferring the enzymatically hydrolyzed material into a co-distillation apparatus, adding water to submerge the material, adding ethanol, adjusting the pH to 4-5 with citric acid, and then distilling.

9. The efficient extraction method according to claim 8, characterized in that, The amount of ethanol added is 8-12% of the weight of fresh lemongrass; After adding ethanol, 0.01~0.05% w / v of defoamer is also added, calculated based on the total volume of the system.

10. The efficient extraction method according to claim 8, characterized in that, The total distillation time is 2 to 2.5 hours.

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