A method for preparing chicory flavor by combining fermentation and maillard reaction technology

Abstract

The application discloses a method for preparing chicory flavor by combining fermentation and Maillard reaction technology, and belongs to the technical field of tobacco flavor. The existing preparation methods of the chicory tobacco flavor mostly adopt single strain fermentation, and have problems of low conversion efficiency of aroma components, unobvious characteristic flavor and the like. The method comprises the following steps: taking chicory roots as raw materials, carrying out ultramicro pulverization, inoculating Bacillus pumilus and Lactobacillus plantarum composite strains to carry out liquid submerged fermentation, adding a Maillard reaction promoter into the fermentation product to carry out Maillard reaction, and finally carrying out centrifugation, filtration, concentration and drying to obtain the chicory flavor. In the method, the composite strain fermentation realizes metabolic complementation of cell wall degradation and accumulation of aroma precursor substances, the Maillard reaction promoter directionally generates characteristic aroma components such as 2-methylbutanal and 3-methylbutanal, and the chicory flavor is endowed with unique caramel sweet and cocoa / nutty fragrance. The obtained chicory flavor is suitable for cigarette flavoring, enriches tobacco aroma levels, and improves sensory quality.

Description

Technical Field

[0001] This invention belongs to the field of tobacco flavoring technology, specifically relating to a method for preparing chicory flavoring by combining fermentation and Maillard reaction technology. Background Technology

[0002] endive( Chicory intybus L. The roots are rich in active ingredients such as inulin, chlorogenic acid and chicoric acid. After heat treatment, they can produce flavor substances such as caramel sweetness and roasting aroma, making them a raw material for preparing natural tobacco flavorings.

[0003] CN107227204A discloses the use of Bacillus subtilis (… Bacillus subtilis CN107227317A further discloses a method for preparing tobacco flavoring by fermenting chicory roots, specifically using Bacillus pumilus (…). Bacillus pumilus The above methods all employ a single microbial strain for fermentation to prepare tobacco flavorings. However, the conversion efficiency of aroma-producing components is limited.

[0004] In recent years, the technology of combining fermentation with Maillard reaction to prepare tobacco flavorings has gradually attracted attention. For example, CN117946809B discloses a technical scheme for preparing tobacco flavorings by using *Candida utilis* and *Bacillus cereus* to ferment licorice together, and then adding fructose to carry out Maillard reaction. Since the main active components of licorice are glycyrrhizic acid and glycyrrhizin, they are easily metabolized and transformed by lactic acid bacteria and other organisms during fermentation. Their decomposition products, such as glycyrrhetinic acid, have strong physiological activity, and their application in cigarettes may bring uncontrollable pharmacological risks.

[0005] Therefore, developing a tobacco flavoring with higher raw material safety and purer flavor characteristics has broad market value. Summary of the Invention

[0006] The present invention aims to overcome the shortcomings of the prior art and provide a method for preparing chicory flavoring by combining fermentation and Maillard reaction technology. Through the synergistic use of compound microbial fermentation and Maillard reaction, caramel sweetness and cocoa / nut aroma are generated in a targeted manner.

[0007] In a first aspect, the present invention provides a method for preparing chicory flavoring by combining fermentation and Maillard reaction techniques, comprising the following steps: S1. Raw material pretreatment: Chicory root is used as raw material, and is washed, dried and crushed to obtain chicory root powder; S2. Compound microbial fermentation: Water is added to chicory root powder, and a compound microbial culture of Bacillus pumilus and Lactobacillus plantarum is inoculated for liquid deep fermentation. The fermentation temperature is 30-40℃, the fermentation time is 36-60 hours, and the pH is controlled at 5.5-6.5 to obtain the fermentation product. S3. Maillard reaction: Add Maillard reaction promoter to fermentation product, and then carry out Maillard reaction at 90-120℃ for 2-6 hours to obtain Maillard reaction product; S4. Post-processing: The Maillard reaction product is centrifuged, filtered, concentrated, and dried to obtain the chicory flavoring.

[0008] Preferably, in step S1, the pulverization is ultrafine pulverization, with a particle size of 45-150 μm. Chicory root powder within this particle size range is beneficial for sufficient contact between microorganisms and improved fermentation efficiency; if the particle size is too large, fermentation will be insufficient; if the particle size is too small, solid-liquid separation in the filtration step will be difficult.

[0009] Preferably, in step S2, the mass ratio of *Bacillus pumilus* to *Lactobacillus plantarum* is 1:10-10:1, more preferably 1:5-5:1; further preferably 1:2-2:1; and most preferably 1:1. When the mass ratio is less than 1:10, the number of *Bacillus pumilus* is insufficient, the cell wall is not sufficiently disrupted, and the release of bound aroma precursors is limited; when the mass ratio is greater than 10:1, the number of *Lactobacillus plantarum* is insufficient, making it difficult to establish an acidic microenvironment, and the accumulation efficiency of aroma precursors decreases; when the two are combined in a 1:1 ratio, the synergistic effect is most significant.

[0010] Preferably, the *Bacillus pumilus* Bacillus pumilus It is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC NO.3411; deposit date: November 5, 2009.

[0011] Preferably, the *Lactobacillus plantarum* Lactobacillus plantarum It is deposited at the China General Microbiological Culture Collection Center (CGMCC); accession number: CGMCC NO.24791; deposit date: April 27, 2022.

[0012] Preferably, in step S2, the inoculum amount for fermentation is 1%-5% of the chicory root powder mass, and the stirring speed is 100-300 rpm. When the inoculum amount is less than 1%, fermentation starts slowly and production efficiency is low; when the inoculum amount is more than 5%, excessive cell growth may produce undesirable metabolites.

[0013] Preferably, in step S2, the fermentation adopts a liquid submerged fermentation method, and the liquid volume of the fermenter is 60%-80%. This liquid volume ensures sufficient dissolved oxygen levels while preventing foam overflow during fermentation. Sterile air is introduced during fermentation at a rate of 0.5-2.0 vvm to maintain dissolved oxygen at no less than 20%. During fermentation, food-grade sodium carbonate solution is added to maintain the pH at 5.5-6.5.

[0014] Preferably, in step S3, the Maillard reaction promoter is selected from at least two of leucine, isoleucine, and fructose. Leucine and isoleucine can generate 2-methylbutyraldehyde and 3-methylbutyraldehyde in the Maillard reaction, imparting a cocoa / nut flavor to chicory flavor; fructose, as a reducing sugar, can provide a carbonyl group to participate in the Maillard reaction, enhancing the caramel sweetness.

[0015] Preferably, the amount of leucine and isoleucine added is 0.1%-2.0% of the mass of the fermentation product, and the amount of fructose added is 1.0%-5.0% of the mass of the fermentation product. When the amount added is below the lower limit, the targeted aroma enhancement effect is not significant; when the amount added is above the upper limit, it may produce unpleasant flavors or increase production costs.

[0016] More preferably, the Maillard reaction promoter is a combination of leucine and isoleucine, with each added at an amount of 0.1%-2.0% of the fermentation product mass, and the mass ratio of leucine to isoleucine is 1:2-2:1, with 1:1 being the most preferred.

[0017] In another preferred embodiment, the Maillard reaction promoter is a combination of leucine, isoleucine and fructose, wherein the mass ratio of leucine to isoleucine is 1:1, the amount of each is 0.2%-2.0% of the mass of the fermentation product, and the amount of fructose is 1.0%-4.0% of the mass of the fermentation product.

[0018] Preferably, in step S3, the pH of the Maillard reaction is controlled between 6.5 and 7.5. During the reaction, a food-grade phosphate buffer (such as a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pair) or a citrate buffer can be added as a pH buffer. This pH range is beneficial for the Maillard reaction and avoids the degradation of aroma components under excessively acidic or alkaline conditions.

[0019] Preferably, in step S3, the Maillard reaction temperature is 90-120°C. Below 90°C, the Maillard reaction rate is too low, resulting in insufficient formation of aroma components; above 120°C, a burnt flavor is easily produced, affecting the quality of the chicory flavoring.

[0020] Preferably, in step S3, the Maillard reaction takes 2-6 hours. If the reaction time is less than 2 hours, the Maillard reaction is incomplete, resulting in insufficient formation of aroma components; if the reaction time is more than 6 hours, production efficiency decreases, and undesirable byproducts may be generated.

[0021] Preferably, steps S2 and S3 can be carried out in situ in the same reactor. After fermentation, there is no need to separate the cells. First, add food-grade phosphate buffer to adjust the pH of the system to 6.5-7.5, and then directly heat to the Maillard reaction temperature. The one-pot process simplifies the operation steps, reduces equipment investment, and avoids the loss of intermediate products during the separation process.

[0022] Furthermore, in step S2, the liquid submerged fermentation is carried out in a fermenter. After fermentation, the fermentation product is heated to the Maillard reaction temperature by means of tank jacket heating or direct steam introduction. This operation mode enables continuous production of fermentation and Maillard reaction.

[0023] Preferably, in step S4, the concentration is performed under vacuum, with a concentration temperature of 50-70°C, a vacuum degree of -0.08 to -0.095 MPa, and concentration until the solid content is 40%-60%. Vacuum concentration can avoid the loss of heat-sensitive aroma components.

[0024] Preferably, in step S4, the drying is either spray drying or freeze drying. Spray drying is suitable for large-scale production, with an inlet air temperature of 160-200℃ and an outlet air temperature of 80-100℃; freeze drying is suitable for the preparation of high value-added products, involving pre-freezing at -40℃ for 4-8 hours, followed by freeze drying under a vacuum of less than 10Pa for 24-72 hours. This operation can maximize the retention of aroma components.

[0025] Furthermore, the method further includes step S5: mixing the chicory flavoring obtained in step S4 with a food-grade anti-caking agent, wherein the food-grade anti-caking agent is selected from at least one of silica, tricalcium phosphate, and microcrystalline cellulose, and the addition amount is 0.5%-2.0% of the chicory flavoring's mass. The specifications of the food-grade anti-caking agent all comply with the relevant national food safety standards. The addition of the food-grade anti-caking agent can prevent the powdered chicory flavoring from clumping during storage, improving its flowability and storage stability.

[0026] In a second aspect, the present invention provides a chicory flavoring prepared by the method described in the first aspect.

[0027] The chicory flavoring contains caramel-sweet and cocoa / nut-characteristic aroma components such as pyrazines, furans, 2-methylbutyraldehyde, and 3-methylbutyraldehyde.

[0028] Preferably, the chicory flavoring is used to flavor cigarettes, and the amount added is 0.01%-0.5% of the tobacco shreds. When the amount added is less than 0.01%, the flavoring effect is not significant; when the amount added is more than 0.5%, it may mask the natural aroma of tobacco and affect the harmony of the cigarette.

[0029] The present invention has the following beneficial effects: (1) This invention combines Bacillus pumilus and Lactobacillus plantarum for the fermentation of chicory roots to prepare tobacco flavorings. The two strains have complementary metabolic functions: Bacillus pumilus secretes extracellular enzymes to break down cell walls and release bound aroma precursors; Lactobacillus plantarum produces acid to create an acidic microenvironment, promoting the accumulation of aroma precursors. The synergistic effect is most significant when the two are combined at a mass ratio of 1:1. Compared with single-strain fermentation, the content of key aroma components is increased by more than 50%, and the total sensory evaluation score is increased from 61.0 points to 70.0 points.

[0030] (2) This invention organically combines fermentation with the Maillard reaction, and uses at least two of leucine, isoleucine, and fructose as Maillard reaction promoters to directionally generate characteristic aroma components such as 2-methylbutyraldehyde and 3-methylbutyraldehyde, giving chicory flavor a unique caramel sweet aroma and cocoa / nut complex aroma. Experiments show that after adding leucine and isoleucine, the cocoa / nut aroma score increased from 1.5 points to 5.0 points, and after further adding fructose, the HDMF content increased from 105.3 μg / g to 119.3 μg / g.

[0031] (3) The present invention can adopt a one-pot process. After the fermentation is completed, the cells are not separated. The pH is directly adjusted and the temperature is raised to carry out the Maillard reaction. While ensuring the quality of chicory flavor, the operation steps are simplified and it is suitable for industrial continuous production.

[0032] (4) When the chicory flavoring prepared in this invention is used to flavor cigarettes, the amount added is 0.01%-0.5% of the tobacco shreds. It can significantly enhance the caramel sweet aroma and cocoa / nut aroma of cigarettes, enrich the layers of tobacco aroma, and improve the taste comfort. Detailed Implementation

[0033] To more clearly illustrate the present invention, the following description, in conjunction with embodiments and comparative examples, further clarifies the invention. Those skilled in the art should understand that the following description represents preferred embodiments of the invention, but the invention is not limited to these embodiments.

[0034] Testing methods and evaluation standards The chicory flavorings obtained in the following examples and comparative examples were all tested and evaluated using the following methods: 1. Quantitative Detection of Key Aroma Components by GC-MS Key aroma components in chicory flavor were quantitatively analyzed using gas chromatography-mass spectrometry (GC-MS).

[0035] (1) Sample pretreatment: Accurately weigh 0.5 g of chicory flavoring sample and place it in one end of a simultaneous distillation extraction apparatus. Add 100 mL of distilled water and 30 mL of dichloromethane to the other end. Heat and reflux for 1.5 hours to extract the organic phase. Add anhydrous sodium sulfate and dry overnight. After filtration, concentrate to 1 mL using a rotary evaporator. Add internal standard (phenethyl acetate), filter through a 0.45 μm organic filter membrane, and then analyze by GC-MS.

[0036] (2) Chromatographic conditions: The chromatographic column was a DB-5MS capillary column (30m×0.25mm×0.25μm); the temperature program was 40℃ for 2 min, then increased to 250℃ at 5℃ / min and held for 10 min; the carrier gas was helium with a flow rate of 1.0 mL / min; the injection port temperature was 250℃; the injection volume was 1 μL, and the injection was splitless.

[0037] (3) Mass spectrometry conditions: EI ion source, electron energy 70eV, ion source temperature 230℃, quadrupole temperature 150℃, full scan mode, scan range m / z 30-450.

[0038] (4) Quantitative methods: The internal standard curve method was used to quantify the following key aroma components: 2-methylbutyraldehyde, 3-methylbutyraldehyde, 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, 2-ethyl-3,5-dimethylpyrazine, and 4-hydroxy-2,5-dimethyl-3(2H)-furanone (HDMF). The quantitative results are expressed in μg / g.

[0039] The "Total Pyrazines" in the table is the sum of 2,5-dimethylpyrazine, 2,3,5-trimethylpyrazine, and 2-ethyl-3,5-dimethylpyrazine.

[0040] 2. Sensory evaluation methods and scoring criteria (1) Cigarette flavoring and sample preparation The chicory flavoring obtained from each example and comparative example was evenly sprayed onto blank cigarette tobacco at a ratio of 0.05% of the tobacco shreds by a micro-sprayer, and then rolled into experimental cigarettes. Cigarettes rolled from blank tobacco without any added flavoring were used as a blank control.

[0041] (2) Evaluation methods and standards An evaluation panel of 10 evaluators with more than five years of experience in tobacco sensory evaluation was formed. The evaluation was conducted in a dedicated evaluation room that was well-ventilated, odorless, with a temperature of 22±2℃ and a relative humidity of 60±5%.

[0042] Referring to YC / T 415—2011 "Sensory Evaluation Method for Tobacco Products", five indicators of cigarettes were scored: aroma (10 points), harmony (6 points), off-flavors (12 points), irritation (20 points), and aftertaste (25 points), with a total sensory evaluation score of 73 points. The average score of 10 evaluators was taken as the final score.

[0043] The scores for each indicator of the blank control cigarette were as follows: aroma 5.0 points, harmony 3.0 points, off-flavors 7.0 points, irritation 16.0 points, aftertaste 19.0 points, for a total of 50.0 points.

[0044] Meanwhile, four characteristic attributes—caramel sweetness, cocoa / nut aroma, smoke smoothness, and aftertaste—were individually scored from 1 to 5 points, with higher scores indicating a more pronounced attribute. The blank control cigarettes received a score of 1.0 for each characteristic attribute.

[0045] Examples 1-5: Screening of Composite Microbial Strains by Mass Ratio Example 1 S2. Compound Microbial Fermentation: Take 100g of chicory root powder, add 800g of water and mix well, then transfer to a 2L small fermenter. Inoculate with a compound microbial culture (Bacillus pumilus CGMCC NO.3411 and Lactobacillus plantarum CGMCC NO.24791 in a mass ratio of 1:10) at 3% of the chicory root powder mass for liquid submerged fermentation. Fermentation conditions: temperature 35℃, stirring speed 200rpm, liquid volume 70%, sterile air purging at a rate of 1.0 vvm, maintaining dissolved oxygen at no less than 20%. During fermentation, add food-grade sodium carbonate solution to maintain pH at 6.0±0.3. Fermentation time is 48 hours, yielding the fermentation product. The fermentation endpoint is determined by the pH value of the fermentation product. When the pH value of the fermentation product changes by no more than 0.1 within 2 hours, it indicates that the metabolic activity of the compound microbial culture has basically stopped, and fermentation is considered complete.

[0046] S3. Maillard Reaction: Maillard reaction promoters, namely leucine and isoleucine, were added to the fermentation product at a ratio of 1.0% of the fermentation product mass, with a mass ratio of leucine to isoleucine of 1:1. Subsequently, a disodium hydrogen phosphate-sodium dihydrogen phosphate buffer pair was added to adjust the pH of the system to 7.0. Finally, the system was heated to 105°C and reacted for 4 hours to obtain the Maillard reaction product. The endpoint of the Maillard reaction was determined by the color of the reaction solution; the endpoint was identified when the Maillard reaction product changed from light yellow to a stable reddish-brown.

[0047] S4. Post-processing: The Maillard reaction product was centrifuged at 4000 rpm for 20 minutes, and the supernatant was filtered. The filtrate was concentrated under vacuum at 60℃ and -0.09 MPa until the solid content was 50%. The concentrate was spray-dried at an inlet air temperature of 180℃ and an outlet air temperature of 90℃ to obtain powdered chicory flavoring.

[0048] Example 2 The method is the same as in Example 1, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is set to 1:2. The remaining steps and conditions are the same as in Example 1.

[0049] Example 3 The method is the same as in Example 1, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is set to 1:1. The remaining steps and conditions are the same as in Example 1.

[0050] Example 4 The method is the same as in Example 1, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is set to 2:1. The remaining steps and conditions are the same as in Example 1.

[0051] Example 5 The method is the same as in Example 1, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is set to 10:1. The remaining steps and conditions are the same as in Example 1.

[0052] The test results of Examples 1-5 are summarized in Table 1.

[0053] Table 1. Effects of different strains on key aroma components and sensory quality of chicory flavoring.

[0054] As shown in Table 1, in Example 3, when the mass ratio of Bacillus pumilus to Lactobacillus plantarum was 1:1, the total content of the six key aroma components reached the highest value of 292.6 μg / g, and the total sensory evaluation score reached the highest value of 70 points. The caramel sweet aroma and cocoa / nut aroma both reached 5.0 points, indicating the most significant synergistic effect. When the mass ratio of the two strains deviated from 1:1, all indicators showed a downward trend. The purpose of this invention can still be achieved in the range of 1:10 to 10:1, but the effect is better in the range of 1:2 to 2:1, with 1:1 being the optimal ratio.

[0055] Examples 6-7: Screening of different Maillard reaction promoter combinations (based on Example 3).

[0056] Example 6 The method is the same as in Example 3, except that in step S3, the merard reaction promoter is 1.0% leucine and 2.5% fructose, and isoleucine is not added. The remaining steps and conditions are the same as in Example 3.

[0057] Example 7 The method is the same as in Example 3, except that the merard reaction promoter in step S3 is 1.0% leucine, 1.0% isoleucine, and 2.5% fructose in a mass ratio of 1:1. The remaining steps and conditions are the same as in Example 3.

[0058] The test results of Example 3 (leucine + isoleucine), Example 6 (leucine + fructose), and Example 7 (leucine + isoleucine + fructose) are summarized in Table 2.

[0059] Table 2. Effects of Maillard reaction promoters on key aroma components and sensory quality of chicory flavoring.

[0060] As shown in Table 2, in Example 6, which only added leucine and fructose, the 2-methylbutyraldehyde content was only 12.8 μg / g, the cocoa / nut aroma score was only 2.5 points, and the total sensory evaluation score was 63.0 points. Although the caramel sweet aroma was present, the cocoa / nut aroma was incomplete, and the overall aroma coordination was obviously insufficient.

[0061] In Example 3, the addition of leucine and isoleucine increased the content of 2-methylbutyraldehyde and 3-methylbutyraldehyde to 45.2 μg / g and 68.7 μg / g, respectively. The cocoa / nut aroma score increased from 2.5 to 5.0, and the total sensory evaluation score increased from 63.0 to 70.0. This indicates that the combination of leucine and isoleucine has a synergistic effect, which is superior to that of single-branched amino acids.

[0062] In Example 7, the HDMF content increased from 105.3 μg / g to 119.3 μg / g, the aftertaste sweetness increased from 4.5 to 5.0, and the total sensory evaluation score further increased to 71.0, indicating that fructose can significantly enhance the caramel aroma and aftertaste sweetness.

[0063] Examples 8-9: Screening of different Maillard reaction conditions (based on Example 3).

[0064] Example 8 The method is the same as in Example 3, except that the Maillard reaction conditions in step S3 are 90°C for 2 hours. The remaining steps and conditions are the same as in Example 3.

[0065] Example 9 The method of Example 3 is followed, except that the Maillard reaction conditions in step S3 are 120°C for 6 hours. The remaining steps and conditions are the same as in Example 3.

[0066] The test results of Example 3 (reaction at 105℃ for 4 hours), Example 8 (reaction at 90℃ for 2 hours), and Example 9 (reaction at 120℃ for 6 hours) are summarized in Table 3.

[0067] Table 3. Effects of different Maillard reaction conditions on the quality of chicory flavor.

[0068] As shown in Table 3, in Example 8, the Maillard reaction was less pronounced after reacting at 90°C for 2 hours. The contents of 2-methylbutyraldehyde, 3-methylbutyraldehyde, total pyrazines, and HDMF were 32.6 μg / g, 48.2 μg / g, 52.8 μg / g, and 72.5 μg / g, respectively, all significantly lower than those in Example 3. The scores for caramel sweetness and cocoa / nut aroma were only 3.5 and 3.0, respectively, with a total sensory evaluation score of 62.5. This indicates that the aroma-producing components were insufficiently generated under low-temperature and short-time conditions, resulting in a weak characteristic aroma.

[0069] In Example 9, the reaction was carried out at 120°C for 6 hours. Although the content of aroma components increased further, the score of off-odors decreased from 11.0 to 9.0, and the score of irritation decreased from 19.5 to 17.5. The total sensory evaluation score actually decreased to 61.0, even lower than the 90°C / 2-hour condition. This is because the high temperature and long-term reaction produced a burnt smell and unpleasant irritating odor, which seriously affected the sensory quality.

[0070] Examples 10-11: Screening of different particle sizes Example 10 The method is the same as in Example 3, except that the particle size in step S1 is 45 μm. The remaining steps and conditions are the same as in Example 3.

[0071] Example 11 The method is the same as in Example 3, except that the particle size is 150 μm in step S1. The remaining steps and conditions are the same as in Example 3.

[0072] The results showed that the total sensory evaluation score of chicory flavoring in Example 10 was 70.0 points, which was comparable to that in Example 3, but the solid-liquid separation of the 45 μm particle size sample was difficult in the filtration step; the total sensory evaluation score of chicory flavoring in Example 11 dropped to 68.2 points, because the cell wall was not broken down sufficiently when the particle size was larger, resulting in insufficient release of aroma precursor substances.

[0073] Examples 12-14: Screening of different inoculum amounts for compound microbial strains Example 12 The method is the same as in Example 3, except that the amount of compound microbial inoculum in step S2 is 1% of the mass of chicory root powder. The remaining steps and conditions are the same as in Example 3.

[0074] Example 13 The method is the same as in Example 3, except that the amount of compound microbial inoculum in step S2 is 5% of the mass of chicory root powder. The remaining steps and conditions are the same as in Example 3.

[0075] Example 14 The method is the same as in Example 3, except that the amount of compound microbial inoculum used in step S2 is 8% of the mass of chicory root powder. The remaining steps and conditions are the same as in Example 3.

[0076] The results showed that fermentation proceeded normally when the inoculum concentration was 1%, with a total sensory evaluation score of 66.0. A concentration of 5% yielded better results, with a total sensory evaluation score of 68.7, similar to that of Example 3. Increasing the inoculum concentration to 8% resulted in a slight unpleasant odor due to excessive microbial growth, causing the total sensory evaluation score to drop to 63.0. Considering all factors, the optimal inoculum concentration is 1%-5% of the chicory root powder mass.

[0077] Example 15 The method is the same as in Example 3, except that sterile air is not introduced (aeration rate 0 vvm) and pH adjustment is not performed during the fermentation process in step S2 (no food-grade sodium carbonate solution is added). The remaining steps and conditions are the same as in Example 3.

[0078] The results showed that without the introduction of sterile air and pH adjustment during fermentation, the fermentation efficiency decreased, the total sensory evaluation score was only 55.5 points, and the chicory flavoring had a sour and rancid odor. In Example 3, sterile air was introduced during fermentation (airflow rate 1.0 vvm, maintaining dissolved oxygen at no less than 20%), and food-grade sodium carbonate solution was added to maintain the pH at 6.0 ± 0.3. The resulting chicory flavoring achieved a total sensory evaluation score of 70.0 points, with a pure and full aroma. This indicates that maintaining suitable dissolved oxygen levels and pH conditions is crucial for ensuring the quality of the fermentation product.

[0079] Example 16 The method is the same as in Example 3, except that the drying method in step S4 is freeze drying. Specifically, the concentrate is pre-frozen at -40°C for 6 hours and then freeze-dried under a vacuum of less than 10 Pa for 48 hours. The remaining steps and conditions are the same as in Example 3.

[0080] The results showed that GC-MS analysis revealed the following contents: 2-methylbutyraldehyde content was 47.5 μg / g, 3-methylbutyraldehyde content was 72.3 μg / g, total pyrazine content was 76.8 μg / g, and HDMF content was 112.5 μg / g. The sensory evaluation score was 71.0 points, with 5.0 points for caramel sweetness, 5.0 points for cocoa / nut aroma, 5.0 points for smoke smoothness, and 5.0 points for aftertaste. The total score was slightly higher than that of the chicory flavoring in Example 3, indicating a fresher and more natural aroma. This demonstrates that freeze-drying can better preserve heat-sensitive aroma components, making it suitable for the preparation of high-value-added products.

[0081] Example 17 The method is the same as in Example 3, except that step S5 is added, which involves mixing the chicory flavoring obtained in step S4 with a food-grade anti-caking agent. The remaining steps and conditions are the same as in Example 3.

[0082] The results showed that when 1.0% (by weight) of food-grade silica was added to the obtained chicory flavoring, mixed thoroughly, and stored at 40°C and 75% relative humidity for 30 days, the powdered chicory flavoring did not clump and maintained good flowability. In contrast, chicory flavoring without the added food-grade anti-caking agent showed significant clumping after 15 days under the same conditions. This indicates that adding a food-grade anti-caking agent can effectively improve the storage stability of chicory flavoring.

[0083] Comparative Example 1 The method of Example 3 is followed, except that step S3 is omitted. Specifically, after fermentation, the Maillard reaction is not carried out, and the fermentation product is directly centrifuged, filtered, concentrated and spray-dried. The remaining steps and conditions are the same as in Example 3.

[0084] The results showed that the obtained chicory flavoring contained 8.2 μg / g of 2-methylbutyraldehyde, 11.5 μg / g of 3-methylbutyraldehyde, 18.6 μg / g of total pyrazines, and 22.3 μg / g of HDMF, as determined by GC-MS. The total sensory evaluation score was 54.5, with a caramel sweet aroma score of 1.5 and a cocoa / nut aroma score of 1.0. Comparative Example 1 lacked characteristic aromas such as caramel sweetness and cocoa / nut aroma, exhibiting a thin fragrance, indicating that the Maillard reaction is a necessary step in generating characteristic aromas.

[0085] Comparative Example 2 The method of Example 3 is followed, except that step S2 is omitted. Specifically, chicory root powder is directly mixed with water without inoculating any microorganisms. After stirring for 48 hours under the same conditions, an equal amount of Maillard reaction promoter is directly added to carry out the Maillard reaction and post-treatment. The remaining steps and conditions are the same as in Example 3.

[0086] The results showed that the obtained chicory flavoring contained 10.5 μg / g of 2-methylbutyraldehyde, 15.2 μg / g of 3-methylbutyraldehyde, 22.5 μg / g of total pyrazines, and 35.8 μg / g of HDMF, as determined by GC-MS. The total sensory evaluation score was 52.0, with a caramel sweetness score of 1.5 and a cocoa / nut aroma score of 1.0. The aroma was thin, dominated by burnt bitterness and raw green notes, indicating that fermentation was key to the release and accumulation of aroma precursors, exhibiting a synergistic effect with the Maillard reaction.

[0087] Comparative Example 3 The method of Example 3 is followed, except that the order of steps S2 and S3 is reversed. First, chicory root powder is mixed with water and then subjected to Maillard reaction (under the same conditions as in Example 3). After the reaction is completed, it is cooled to 35°C, and then an equal amount of compound bacteria is inoculated for fermentation for 48 hours. The remaining steps and conditions are the same as in Example 3.

[0088] The results showed that the obtained chicory flavoring had a total sensory evaluation score of 47.5 points, exhibiting a distinct burnt and bitter taste and an unpleasant fermented odor, making the aroma completely unacceptable. This indicates that the sequence of fermentation followed by Maillard reaction is technically significant.

[0089] Comparative Examples 4-5: Comparison between single-strain and multi-strain cultures Comparative Example 4 The method is the same as in Example 3, except that in step S2, only Bacillus pumilus CGMCC NO.3411 is inoculated, and the inoculation amount is 3%. The remaining steps and conditions are the same as in Example 3.

[0090] Comparative Example 5 The method of Example 3 is followed, except that in step S2 only *Lactobacillus plantarum* CGMCC NO.24791 is inoculated, and the inoculation amount is 3%. The remaining steps and conditions are the same as in Example 3.

[0091] The results of Comparative Example 4, Comparative Example 5 and Example 3 are summarized in Table 4.

[0092] Table 4 Comparison of fermentation effects between single-strain and multi-strain cultures

[0093] Table 4 shows that the content of key aroma components and sensory evaluation scores of single-strain fermentation products were significantly lower than those of multi-strain fermentation. Comparative Example 4 only added Bacillus pumilus, which is mainly responsible for degrading cell walls and releasing bound precursors, resulting in a higher content of pyrazine compounds in its fermentation products. In contrast, Comparative Example 5 only added Lactobacillus plantarum, which can create an acidic environment and promote the accumulation of aroma precursor substances, thus resulting in a higher HDMF content in chicory flavoring. In Example 3, when Bacillus pumilus and Lactobacillus plantarum were combined at a mass ratio of 1:1, metabolic complementarity and synergistic effects were achieved, and the total sensory evaluation scores increased from 61.0 and 58.5 to 70.0, respectively.

[0094] Comparative Examples 6-7: Comparison of Different Maillard Reaction Promoters Comparative Example 6 The method is the same as in Example 3, except that no accelerator is added in step S3. The remaining steps and conditions are the same as in Example 3.

[0095] Comparative Example 7 The method is the same as in Example 3, except that in step S3, leucine and isoleucine are replaced with 1.0% glycine + 1.0% alanine. The remaining steps and conditions are the same as in Example 3.

[0096] The results of Comparative Example 6, Comparative Example 7 and Example 3 are summarized in Table 5.

[0097] Table 5. Effects of different promoters on key aroma components and sensory quality of chicory flavoring

[0098] As shown in Table 5, in Comparative Example 6 without the addition of an accelerator, the contents of 2-methylbutyraldehyde and 3-methylbutyraldehyde were extremely low, and the caramel and cocoa / nut aromas could not be effectively generated. In Comparative Example 7, after replacing leucine and isoleucine with glycine and alanine, a certain caramel aroma was generated, but the contents of 2-methylbutyraldehyde and 3-methylbutyraldehyde did not increase significantly, and the cocoa / nut aroma score was only 1.5. This is because glycine and alanine do not have a branched structure and cannot generate 2-methylbutyraldehyde and 3-methylbutyraldehyde through the Streck degradation reaction. This indicates that the specific accelerator selected in this invention has irreplaceable technical effects.

[0099] Comparative Example 8 The method of Example 3 is the same as in Example 3, except that the pH of the system is not adjusted in step S3 (the pH is about 5.8 at this time), and the temperature is directly raised to 105°C to carry out the Maillard reaction. The remaining steps and conditions are the same as in Example 3.

[0100] The results showed that the chicory flavoring contained 32.5 μg / g of 2-methylbutyraldehyde, 48.2 μg / g of 3-methylbutyraldehyde, 55.6 μg / g of total pyrazines, and 72.3 μg / g of HDMF. The sensory evaluation score was 61.0 points. The obtained chicory flavoring was too dark in color and had a distinct burnt, bitter, and sour taste. This indicates that adjusting the pH of the system to 6.5-7.5 before carrying out the Maillard reaction is a crucial step in ensuring the quality of the chicory flavoring.

[0101] Comparative Examples 9-10: Comparison of Compound Microbial Cultures with Quality Ratios Exceeding the Range Comparative Example 9 The method of Example 3 is the same as that of Example 3, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is 1:15. The remaining steps and conditions are the same as those of Example 3.

[0102] Comparative Example 10 The method of Example 3 is the same as that of Example 3, except that the mass ratio of Bacillus pumilus to Lactobacillus plantarum in step S2 is 15:1, and the rest of the steps and conditions are the same as those of Example 3.

[0103] The results of Comparative Examples 9 and 10 showed that when the mass ratio of Bacillus pumilus to Lactobacillus plantarum in Comparative Example 9 was 1:15, the total sensory evaluation score was 57.0 points, the caramel sweet aroma score was 2.0 points, the cocoa / nut aroma score was 1.5 points, indicating insufficient fermentation and a weak aroma; when the mass ratio of Bacillus pumilus to Lactobacillus plantarum in Comparative Example 10 was 15:1, the total sensory evaluation score was 55.5 points, with a noticeable bitter taste.

[0104] Comparative Examples 11-12: Comparison of Maillard Reaction Conditions Exceeding the Range Comparative Example 11 The method of Example 3 is the same, except that the Maillard reaction conditions in step S3 are 130°C for 8 hours. The remaining steps and conditions are the same as in Example 3.

[0105] Comparative Example 12 The method is the same as in Example 3, except that the Maillard reaction conditions in step S3 are 80°C for 1 hour. The remaining steps and conditions are the same as in Example 3.

[0106] The results showed that the chicory flavoring obtained after reacting at 130℃ for 8 hours contained 52.3 μg / g of 2-methylbutyraldehyde, 78.5 μg / g of 3-methylbutyraldehyde, 85.2 μg / g of total pyrazines, and 125.6 μg / g of HDMF, as determined by GC-MS. Despite the high content of aroma-producing components, the total sensory evaluation score was only 55.0 points. It had a distinct burnt and bitter taste, and a blackish color, making it unsuitable for use as a flavoring.

[0107] The chicory flavoring obtained after reacting at 80℃ for 1 hour was analyzed by GC-MS and found to contain 15.8 μg / g of 2-methylbutyraldehyde, 22.5 μg / g of 3-methylbutyraldehyde, 32.5 μg / g of total pyrazines, and 42.8 μg / g of HDMF. The sensory evaluation score was 58.0 points, indicating incomplete Maillard reaction and weak caramel and cocoa / nut aromas.

[0108] This indicates that when the Maillard reaction temperature and time exceed the range of 90-120℃ and 2-6 hours, the quality of chicory flavoring deteriorates significantly or the reaction efficiency is severely insufficient.

[0109] Comparative Example 13 The method described in Example 5 of CN117946809B (licorice fermentation conditions: 50℃ fermentation for 24h, Maillard reaction at 120℃ for 30min) is followed, except that the "Candida utilis and Bacillus cereus" used are replaced with the compound strain of this invention (Bacillus pumilus CGMCC NO.3411 and Lactobacillus plantarum CGMCC NO.24791, mass ratio 1:1). The remaining steps and conditions are the same as in Example 5 of CN117946809B.

[0110] The results showed that under the conditions of fermentation at 50℃ for 24 hours and Maillard reaction at 120℃ for 30 minutes, the GC-MS analysis results of the licorice flavoring prepared in Comparative Example 13 were as follows: total alcohols, esters, and ketones: 218.5 μg / g; total aldehydes, phenolic acids, and fatty acids: 185.3 μg / g; total aroma compounds: 403.8 μg / g. The total sensory evaluation score was 47.0 points, with aroma scoring 6.5 points, harmony scoring 4.0 points, off-odors scoring 7.5 points, irritation scoring 13.0 points, and aftertaste scoring 16.0 points. The main characteristics were a thin aroma, obvious off-odors, strong irritation, and an unpleasant aftertaste.

[0111] The reasons are as follows: (1) The optimal fermentation temperature for Bacillus pumilus and Lactobacillus plantarum is 30-40℃, while the licorice process requires 50℃. At this temperature, the activity of the strains of this invention is inhibited, and the fermentation efficiency is reduced; (2) The content of branched-chain amino acids in licorice is limited, and leucine and isoleucine were not added exogenously in this comparative example, so 2-methylbutyraldehyde and 3-methylbutyraldehyde cannot be generated in a targeted manner; (3) There is no synergistic effect between fungi and bacteria like that between Candida utilis and Bacillus cereus between Bacillus pumilus and Lactobacillus plantarum.

[0112] This indicates that when the compound microbial strain (Bacillus pumilus + Lactobacillus plantarum) of the present invention is directly applied to the licorice fermentation system, the cocoa / nut aroma described in the present invention cannot be obtained due to incompatible process conditions, substrate differences, and lack of exogenous branched-chain amino acid supplementation.

[0113] Comparative Example 14 Following the method in Example 3, the bacterial strains were replaced with Candida utilis and Bacillus cereus.

[0114] The results showed that, under the Maillard reaction conditions of 120℃ for 30 minutes, the GC-MS detection results of the chicory flavoring prepared in Comparative Example 14 were as follows: 2-methylbutyraldehyde was not detected (<0.1 μg / g), 3-methylbutyraldehyde was not detected (<0.1 μg / g), the total pyrazine content was only 6.8 μg / g (characteristic pyrazines such as 2-ethyl-3,5-dimethylpyrazine were not detected), and the HDMF content was 8.5 μg / g. The total sensory evaluation score was 43.0 points, including 5.5 points for aroma, 3.0 points for harmony, 6.5 points for off-odors, 12.0 points for irritation, and 16.0 points for aftertaste.

[0115] This indicates that the combination of Candida utilis and Bacillus cereus on chicory roots cannot generate specific Maillard reaction precursors such as N-fructosylpyroglutamic acid and characteristic substances such as 2-ethyl-3,5-dimethylpyrazine. Furthermore, the content of pyrazines and HDMF is much lower than that in Example 3 of this invention, and the quality of chicory flavor is lower than that of this invention.

[0116] Comparative Example 15 Following the method of Example 5 of CN117946809B, licorice was fermented using a combination of Candida utilis and Bacillus cereus (mass ratio 1:1), followed by the addition of fructose for Maillard reaction. The remaining steps and conditions were the same as in Example 5 of CN117946809B.

[0117] The results showed that the licorice flavoring prepared in Comparative Example 15, as determined by GC-MS, contained furfural, 5-methylfuranaldehyde, 2-acetylfuran, furfuryl alcohol, and other furan compounds, as well as pyrazine compounds such as 2,6-dimethylpyrazine and 2-methylpyrazine. No pyrazine compounds characteristic of cocoa aroma, such as 2-ethyl-3,5-dimethylpyrazine, were detected. Sensory evaluation revealed a distinct caramel sweetness and characteristic licorice aroma, but no cocoa / nuttiness.

[0118] Studies have shown that the main characteristic components of the Maillard reaction products of licorice are furfural, 5-methylfuranaldehyde, 2-acetylfuran, and other furan compounds (accounting for more than 50% of the total volatile components), rather than cocoa / nut aromas. Other literature reports that the main components of fermented licorice flavoring are hexanoic acid, linoleic acid, 2,3-dihydrobenzofuran, indole, etc., which also do not involve cocoa / nut aroma characteristic substances.

[0119] The above results prove that the technical solution of CN117946809B (Candida utilis fermentation of licorice with Bacillus cereus fermentation and fructose Maillard reaction) produces a caramel sweet aroma and licorice characteristic aroma, rather than the caramel sweet aroma and cocoa / nut complex aroma described in this invention.

[0120] Overall Conclusion The above embodiments and comparative examples fully illustrate: (1) This invention utilizes a compound microbial fermentation process involving a specific mass ratio of Bacillus pumilus and Lactobacillus plantarum. The two microbial strains have complementary metabolisms, resulting in a synergistic effect in cell wall degradation and the accumulation of aroma precursors. Compared with single-strain fermentation, the content of key aroma components in the compound microbial fermentation product is increased by more than 50%. The invention achieves its objectives within a mass ratio range of 1:10 to 10:1, with the optimal effect observed at a 1:1 ratio.

[0121] (2) The chicory flavoring preparation method of the present invention organically combines compound microbial fermentation with Maillard reaction. During fermentation, Bacillus pumilus and Lactobacillus plantarum work together to degrade the cell wall of chicory root, releasing and converting Maillard reaction precursors such as small molecule peptides, free amino acids and reducing sugars, providing a rich substrate basis for subsequent Maillard reaction.

[0122] (3) In this invention, leucine and isoleucine are used as Maillard reaction promoters to directionally generate 2-methylbutyraldehyde and 3-methylbutyraldehyde through the Strecker degradation reaction, giving chicory flavor a unique cocoa / nut aroma. In this invention, fructose can be further added as a Maillard reaction promoter to enhance the caramel sweetness and aftertaste of chicory flavor.

[0123] (4) The present invention adopts a one-pot process to ensure the quality of chicory spices while simplifying the operation process and reducing equipment investment, making it suitable for industrial continuous production.

[0124] (5) The chicory flavoring obtained by the present invention is suitable for flavoring cigarettes, which can enhance the caramel sweetness and cocoa / nut aroma of cigarettes, enrich the layers of tobacco aroma, and improve the taste comfort.

[0125] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Any person skilled in the art can make some modifications or alterations to the above-disclosed technical content to create equivalent embodiments without departing from the scope of the present invention. Any simple modifications, equivalent changes and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention shall still fall within the scope of the present invention.

Claims

1. A method for preparing chicory flavoring by combining fermentation and Maillard reaction techniques, characterized in that, Includes the following steps: S1. Raw material pretreatment: Chicory root is used as raw material, and is washed, dried and crushed to obtain chicory root powder; S2. Compound microbial fermentation: Water is added to chicory root powder, and a compound microbial culture of Bacillus pumilus and Lactobacillus plantarum is inoculated for liquid deep fermentation. The fermentation temperature is 30-40℃, the fermentation time is 36-60 hours, and the pH is controlled at 5.5-6.5 to obtain the fermentation product. S3. Maillard reaction: Add Maillard reaction promoter to fermentation product, and then carry out Maillard reaction at 90-120℃ for 2-6 hours to obtain Maillard reaction product; S4. Post-processing: The Maillard reaction product is centrifuged, filtered, concentrated, and dried to obtain the chicory flavoring.

2. The method according to claim 1, characterized in that, In step S2, the mass ratio of Bacillus pumilus to Lactobacillus plantarum is 1:10-10:

1.

3. The method according to claim 1, characterized in that, In step S3, the Maillard reaction promoter is selected from at least two of leucine, isoleucine, and fructose.

4. The method according to claim 3, characterized in that, The Maillard reaction promoter is a combination of leucine and isoleucine, and the mass ratio of leucine to isoleucine is 1:2-2:

1.

5. The method according to claim 1, characterized in that, In step S1, the pulverization is ultrafine pulverization, with a particle size of 45-150 μm.

6. The method according to claim 1, characterized in that, In step S2, the inoculum amount for fermentation is 1%-5% of the mass of chicory root powder, and the stirring speed is 100-300 rpm.

7. The method according to claim 1, characterized in that, In step S2, sterile air is introduced during fermentation at a rate of 0.5-2.0 vvm, and the pH is maintained at 5.5-6.5 by adding food-grade sodium carbonate solution.

8. The method according to claim 1, characterized in that, Steps S2 and S3 are carried out in situ in the same reactor. After fermentation, there is no need to separate the cells. First, add food-grade phosphate buffer to adjust the pH of the system to 6.5-7.5, and then raise the temperature to the Maillard reaction temperature.

9. The method according to claim 1, characterized in that, In step S4, the drying is either spray drying or freeze drying. When spray drying is used, the inlet air temperature is 160-200℃ and the outlet air temperature is 80-100℃. When freeze drying is used, the product is pre-frozen at -40℃ for 4-8 hours and then freeze-dried at a vacuum of less than 10Pa for 24-72 hours.

10. The method according to claim 1, characterized in that, The method also includes step S5: mixing the chicory flavoring obtained in step S4 with a food-grade anti-caking agent, wherein the food-grade anti-caking agent is selected from at least one of silicon dioxide, tricalcium phosphate, and microcrystalline cellulose, and the amount added is 0.5%-2.0% of the mass of the chicory flavoring.

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