A process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms

Abstract

This invention relates to a process for improving the undesirable flavors of stevia based on liquid fermentation of shiitake mushrooms. This process can reduce the bitterness and grassy taste of stevia while retaining its sweetness. A composite culture medium is constructed using a specific ratio of potato flour, carrot flour, and stevia powder, and shiitake mushrooms are inoculated for liquid fermentation. The preparation steps include: dissolving, filtering, and sterilizing the composite culture medium in a water bath, then inoculating shiitake mushroom seed liquid in the logarithmic growth phase, and cultivating it in a shaker at a specific temperature to obtain a fermentation broth; this fermentation broth, after edible treatment, yields the flavor-improved product. This invention achieves the directional conversion of steviol glycosides through fermentation: while efficiently retaining the core sweet component ribobadiol A, it significantly reduces the content of steviol glycosides and ribobadiol C, and significantly enriches ribobadiol F, which has a sweetening effect. The final flavor-improved product shows a 33.29% reduction in bitterness and a 35.98% reduction in grassy taste, with a sweetness retention rate exceeding 88%.

Description

Technical Field

[0001] This invention belongs to the field of food processing technology, specifically relating to a process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms. Background Technology

[0002] With consumers' growing health awareness, low-sugar and low-calorie foods have become an important development direction for the food industry. Natural high-intensity sweeteners, with their unique advantages of being "calorie-free and highly safe," are gradually replacing traditional sucrose and becoming one of the mainstream choices in the market. Stevia, a perennial herbaceous plant of the Asteraceae family, contains steviol glycosides in its leaves that are 200-300 times sweeter than sucrose. Furthermore, it possesses properties such as not participating in human metabolism and not causing a rise in blood sugar, making it widely used in functional beverages, sugar-free pastries, and health foods. It is currently one of the most promising natural sweeteners.

[0003] However, stevia has significant drawbacks in practical applications: its steviol glycoside derivatives (such as steviol glycoside and rebaudioside C) and volatile herbal components often impart unpleasant bitterness, metallic taste, and grassy flavor. These unpleasant flavors become more pronounced with increasing addition amounts, severely limiting the palatability of stevia in finished foods. To overcome this problem, existing technologies mainly focus on three types of methods: first, chemical modification, which uses enzymatic hydrolysis, acetylation, and other methods to modify the structure of steviol glycosides to reduce bitterness; however, this method involves the use of enzymes or chemical reagents, resulting in high costs and the risk of reagent residues; second, flavor masking, which involves adding cyclodextrin, organic acids, or flavorings to mask unpleasant flavors; however, the masking effect is limited, easily introducing new flavor interferences, and increasing the complexity and cost of the formulation; and third, physical separation, which uses chromatography or membrane filtration techniques to enrich the palatable rebaudioside A; however, the related process equipment requires significant investment, has low separation efficiency, and is costly.

[0004] Meanwhile, the liquid fermentation technology for shiitake mushrooms, with its advantages of short fermentation cycle, high activity of metabolites, natural and green nature, and ease of industrial control, is increasingly widely used in food processing. During fermentation, shiitake mushrooms can secrete various active enzymes such as glycosidases and proteases. Glycosidases can specifically hydrolyze glycosidic bonds, while proteases can degrade bitter peptides, thus providing a theoretical possibility for the biotransformation of undesirable flavors in stevia. The potential of this technology has been supported by some research and patents. For example, Myco disclosed in patent US20210267143A1 a method for improving the flavor of plant proteins through deep liquid fermentation using shiitake mushrooms. This technology uses enzymes such as laccase secreted by the mycelium to oxidize, degrade, and biotransform volatile components and undesirable peptides that cause bitterness and beany taste, enhancing the umami and meaty aroma of the product while removing undesirable flavors, significantly improving sensory acceptance. This practice demonstrates that liquid fermentation technology for shiitake mushrooms has the potential to specifically improve the flavor of food ingredients, providing a useful reference for flavor improvement in stevia.

[0005] Currently, some studies have used liquid fermentation technology of shiitake mushrooms to produce functional components such as shiitake polysaccharides. However, the culture media used are mostly conventional raw materials such as glucose, yeast extract, and wheat bran. No studies have yet adopted the above-mentioned flavor improvement ideas to directly use stevia powder as a component of the culture medium. Through the synergistic optimization of the culture medium, strains, and processes, the metabolic fermentation of shiitake mushrooms and the undesirable flavor of stevia can be improved simultaneously.

[0006] Furthermore, existing research on stevia flavor improvement largely focuses on reducing bitterness, lacking a systematic evaluation of overall flavor dynamics and sweetness characteristics. While some methods can alleviate bitterness, they may lead to a loss of sweetness, or fail to clearly define the actual sweetness of fermentation products relative to sucrose or commercial steviol glycosides, making it difficult to precisely control the dosage of improved stevia in food formulations. Therefore, developing a safe, efficient, and naturally green technology for improving undesirable stevia flavors that balances flavor and sweetness has become an important issue facing the food industry. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the present invention aims to provide a natural, green, and efficient process for improving the undesirable flavors of stevia based on shiitake mushroom liquid fermentation. This improved process involves constructing a composite culture medium centered on carrot powder, potato powder, and stevia powder, and employing a specific shiitake mushroom liquid fermentation process. The goal is to significantly reduce the bitterness and grassy taste of stevia while maximizing the preservation of its sweetness. This invention will also clarify the flavor and sweetness characteristics of the fermentation products, providing reliable data support for subsequent food industry applications and promoting the widespread use of stevia in low-sugar foods.

[0008] Various exemplary and non-limiting aspects of the present invention can be summarized as follows:

[0009] This invention provides a process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms, comprising the following steps: (1) Preparation of fermentation medium: Weigh potato flour, carrot flour and stevia powder, add potassium salt and / or magnesium salt according to fermentation requirements, add to ultrapure water and stir evenly, dissolve in water bath, filter, sterilize at high temperature and high pressure to obtain shiitake mushroom fermentation medium; (2) Inoculation of strain: Inoculate the shiitake mushroom strain seed liquid in the logarithmic growth phase into the shiitake mushroom fermentation medium described in step (1) at an inoculation amount of 5% to 20% by volume; (3) Fermentation culture: The inoculated shiitake mushroom fermentation medium was placed in a shaker and cultured at a constant temperature of 20~30℃ for 5~15 days to obtain stevia fermentation liquid samples; (4) Edible treatment: The unstevia fermentation liquid sample was treated to be edible.

[0010] Preferably, in step (1), the amount of potato flour added to the fermentation medium is 4~20g / L, the amount of carrot flour added is 1~10g / L, and the amount of stevia powder added is 2~16g / L.

[0011] More preferably, in step (1), the amount of potato flour added to the fermentation medium is 6~15 g / L, the amount of carrot flour added is 2~8 g / L, and the amount of stevia powder added is 4~12 g / L.

[0012] Preferably, in step (1), the preparation of potato flour, carrot flour and stevia powder can be carried out using conventional processes in the field, including but not limited to self-made raw material powder, such as fresh raw materials being washed, dried, crushed and passed through an 80-120 mesh sieve; or directly purchasing commercial raw material powder that meets food-grade standards (purity ≥95%).

[0013] Preferably, in step (1), the potassium salt includes at least one of potassium chloride, potassium dihydrogen phosphate, and potassium sulfate, and the magnesium salt includes at least one of magnesium chloride and magnesium sulfate.

[0014] Preferably, in step (1), the stirring operation adopts magnetic stirring with a speed of 500~1500r / min and a stirring time of 10~60min; the water bath dissolution temperature is 50~100℃ and the water bath time is 10~30min; the high temperature and high pressure sterilization temperature is 121℃ and the time is 15~40min.

[0015] Further preferred embodiments include a magnetic stirring speed of 700-1000 r / min, a stirring time of 15-35 min, a water bath temperature of 75-100℃, a water bath time of 15-25 min, and a sterilization time of 20-30 min.

[0016] Preferably, in step (1), the filtration operation can be performed using conventional methods known in the art, and the filter medium includes at least one of coarse cotton cloth, coffee filter paper, and 0.8-micron filter.

[0017] Further preferred options include using coarse cotton cloth for filtration.

[0018] Preferably, in step (2), the shiitake mushroom is a strain of the genus Lentinula edodes, including at least one of the following: shiitake mushroom L9608, shiitake mushroom L241-4, shiitake mushroom B08, shiitake mushroom CPCC 400023, shiitake mushroom ACCC 52357, shiitake mushroom Anxiang No. 2 (L18-1), shiitake mushroom L135-1, shiitake mushroom 808, and shiitake mushroom L9015.

[0019] More preferably, the shiitake mushroom is shiitake mushroom ACCC 52357.

[0020] A further preferred method for fermenting shiitake mushrooms is to be implemented according to the following steps: Seed solution preparation: Cultivate shiitake mushrooms to the logarithmic growth phase to prepare a highly active seed solution; Inoculation: Inoculate the seed culture of the strain in the logarithmic growth phase into the fermentation medium at the prescribed inoculation amount; Cultivation: The inoculated culture medium is placed in a shaker and incubated at a constant temperature at the set temperature. After cultivation, the shiitake mushroom fermentation liquid containing flavor-modified stevia components is obtained, which is the stevia fermentation liquid sample.

[0021] Preferably, in step (2), the logarithmic growth phase seed solution can be prepared using conventional methods in the art.

[0022] In a further preferred embodiment, shiitake mushroom agar slants are inoculated onto potato dextrose agar (PDB) medium under sterile conditions and cultured on a shaker at 90-150 r / min for 2-7 days. The resulting culture is the logarithmic growth phase seed culture.

[0023] Further preferred, the shaking speed is 110~140 r / min, and the culture time is 3~6 days.

[0024] Preferably, in step (3), the inoculation amount of the logarithmic growth period seed liquid is 5%~20% (volume percentage), the shaking speed is 90~150 r / min, the shaking culture temperature is 20~30℃, and the culture time is 5~15 days.

[0025] More preferably, the inoculum amount is 7%~15% (volume percentage), the shaker culture temperature is 24~28℃, and the culture time is 5~12 days.

[0026] Preferably, in step (4), the edible treatment of the stevia samples before and after fermentation is carried out using food safety treatment methods known in the art, including pasteurization, ultra-high temperature instantaneous sterilization and other technologies.

[0027] Further preferred methods employ pasteurization, which can kill residual microorganisms in the fermentation broth while preserving the flavor and sweetness characteristics of stevia to the greatest extent.

[0028] The beneficial effects of this invention are as follows: This invention provides a process for improving the undesirable flavors of stevia based on liquid fermentation of shiitake mushrooms. By constructing a composite culture medium of carrot powder, potato powder, and stevia powder, and inoculating it with shiitake mushrooms for liquid fermentation, undesirable flavors such as bitterness and grassiness in stevia are efficiently degraded while retaining its sweetness. This invention is applicable to the development of products, including but not limited to low-sugar beverages, healthy baked goods, and health foods, using stevia as the core sweetener, providing a natural, green, and efficient flavor improvement solution for related industries.

[0029] This invention employs a biotransformation pathway combining a natural composite culture medium with liquid fermentation of shiitake mushrooms. Through the specific metabolism of shiitake mushroom strains, it achieves targeted regulation of steviol glycoside components, providing a safe, efficient, and naturally green new solution for improving the unpleasant flavor of stevia. During fermentation, shiitake mushrooms secrete specific glycosidases that precisely degrade the core bitter components in stevia, such as rebodiin C and steviol glycosides, reducing unpleasant flavor at its source. This avoids the limitations of physical masking methods that rely solely on exogenous components to mask bitterness, which are prone to flavor recurrence. Furthermore, it eliminates the need for exogenous enzymes and chemical matrices required by chemical modification methods, completely eliminating the risk of reagent residues and perfectly aligning with the food industry's "natural and safe" principles. The core requirement is to enrich ribobadiin F, which has a synergistic effect on sweetness enhancement, through fermentation of shiitake mushrooms. Combined with the sweetness-enhancing effect of ribobadiin F, it can further achieve the synergistic optimization of "reducing bitterness" and "preserving and enhancing sweetness" while effectively retaining the core sweet component ribobadiin A (with a retention rate of over 88%). This solves the shortcomings of existing physical separation methods, which can only reduce bitterness by enriching ribobadiin A and are prone to loss of total sweetness or nutrients.

[0030] The improved process parameters provided by this invention are clearly defined (e.g., 5%-20% inoculation amount of shiitake mushrooms, fermentation temperature 20-30℃), the process is simple (compound culture medium preparation → inoculation of strains → shaker culture), and it is highly controllable. It does not rely on special high-end equipment, significantly reducing the technical threshold and production costs. It provides food companies with a path to improve the undesirable flavor of stevia that is technically feasible, cost-effective, and safe for products. It can ensure the flavor compatibility of stevia in low-sugar health foods and give full play to its advantages as a natural sweetener, which can strongly promote the high-quality application and development of stevia in the low-sugar health food industry. Attached Figure Description

[0031] Figure 1 The graph shows the rate of decrease in sensory scores for undesirable flavors after fermentation of the sample in Example 1.

[0032] Figure 2 The graph shows the rate of decrease in sensory scores for undesirable flavors after fermentation in Example 2.

[0033] Figure 3 The graph shows the rate of decrease in sensory scores for undesirable flavors after fermentation of the sample in Example 3.

[0034] Figure 4 The graph shows the rate of decrease in sensory scores for undesirable flavors after fermentation in Example 4.

[0035] Figure 5 This is a graph showing the equivalent sweetness of the samples before and after fermentation relative to sucrose in Example 5.

[0036] Figure 6 The graph shows the equivalent sweetness results of the samples before and after fermentation relative to steviol glycosides in Example 5.

[0037] Figure 7 The graph shows the equivalent sweetness of the samples before and after fermentation relative to sucralose in Example 5.

[0038] Figure 8 The image shows the sensory results of the TDS of the pre-fermentation sample in Example 5.

[0039] Figure 9 The image shows the sensory results of the TDS of the fermented sample in Example 5.

[0040] Figure 10 The above are liquid chromatography results of the steviosides in the samples before and after fermentation in Example 5.

[0041] Figure 11 The graph shows the changes in the content of each steviol glycoside in the samples before and after fermentation in Example 5. Detailed Implementation

[0042] In this invention, the embodiments are described in a clear and concise manner. It should be understood that various combinations, adjustments, or deletions can be made to the embodiments without departing from the essential spirit and scope of this invention. In particular, the proportions, sources, and preparation methods of the components in the composite culture medium can also be appropriately adjusted or replaced.

[0043] In some embodiments, the present invention can be interpreted as excluding any elements or steps that do not substantially affect the improvement of undesirable stevia flavor or the feasibility of the fermentation process. Furthermore, in other embodiments, the present invention can also be interpreted as excluding any non-essential components or process steps not expressly listed herein.

[0044] Although the present invention has been described in detail herein by way of specific embodiments, the scope of the invention is not limited to the details shown in these embodiments. Any modifications, equivalent substitutions, or improvements made within the scope of the inventive concept should be included within the protection scope of the present invention.

[0045] The present invention will be further explained and described below with reference to specific embodiments.

[0046] The shiitake mushroom fermentation medium and stevia fermentation broth samples prepared in the examples were processed to an edible state, and the changes in undesirable flavors and sweetness of stevia before and after fermentation were detected using the following methods: Undesirable flavors are detected by sensory evaluation panel testing or by chemical quantitative analysis to characterize changes in bitter and sweet substances.

[0047] Sensory evaluation was employed: A professionally trained sensory evaluation team conducted blind evaluations of the bitterness and grassy taste of samples before and after fermentation using a 15-point scale (0 points indicating no flavor, 15 points indicating extremely strong flavor). The improvement in palatability was directly reflected by calculating the flavor decline rate. The calculation formula was: Flavor decline rate = (Pre-fermentation score - Post-fermentation score) / Pre-fermentation score × 100%).

[0048] The sweetness change was detected using the equivalent sweetness method: sucrose, commercial steviol glycosides, and commercial sucralose were used as standard sweetener references. A sensory evaluation team matched the sweetness of the test sample with the references, and the concentration of the references at which there was no significant difference in sensory sweetness between the test sample and the references was determined as the equivalent sweetness. This method was used to quantitatively assess the impact of the fermentation process on sweetness.

[0049] Undesirable flavors and changes in sweetness were assessed using the TDS dynamic sensory evaluation method to evaluate the temporal changes in flavor profiles. Sensory evaluation team members continuously recorded the "dominance rate" (i.e. the proportion of flavors perceived as the most prominent at a certain moment) of each flavor attribute in the oral cavity from 0 to 60 seconds. By plotting the flavor dominance rate-time curve, the duration of the dominance of undesirable flavors after fermentation was analyzed to see if the dominance of sweetness was shortened or enhanced, thereby comprehensively verifying the overall effect of flavor improvement from a dynamic perception perspective.

[0050] Undesirable flavor and sweetness changes were analyzed using quantitative chemical methods, including but not limited to high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), ultraviolet-visible spectrophotometry (UV-Vis), thin-layer chromatography (TLC), near-infrared spectroscopy, and fluorescence spectroscopy. Preferably, HPLC was used for quantitative analysis.

[0051] High performance liquid chromatography (HPLC) is used to quantitatively detect changes in the content of various steviol glycosides in samples before and after fermentation.

[0052] The procedure for determining the content of steviol glycosides by high performance liquid chromatography shall be performed in accordance with GB 1886.355-2022.

[0053] The detection of various steviol glycosides includes, but is not limited to, ribobadiin A, steviol glycoside, ribobadiin F, and ribobadiin C.

[0054] Example 1 (1) Preparation and fermentation of composite culture medium: Prepare 1 L of composite culture medium containing 15 g / L potato flour, 8 g / L carrot flour, 12 g / L stevia powder, 0.5 g / L potassium dihydrogen phosphate, and 0.2 g / L magnesium sulfate. Add ultrapure water to 1 L. Stir magnetically at 500 r / min for 10 min, and then dissolve in a water bath at 100℃ for 25 min. Filter the mixed solution through coarse cotton cloth, dispense into 100 mL Erlenmeyer flasks, autoclave at high temperature and pressure for 40 min, and cool to room temperature for later use.

[0055] Under aseptic conditions, 12% (by volume) of the shiitake mushroom ACCC 52357 seed culture in the logarithmic growth phase was inoculated into the above culture medium and fermented in a shaker at 28°C and 150 rpm for 12 days.

[0056] (2) Sample pretreatment and subject screening: After fermentation, the fermentation broth was filtered and pasteurized to serve as the experimental group sample. The control group consisted of unfermented shiitake mushroom culture medium that underwent the same pasteurization treatment. For sensory evaluation, 20 initial subjects were recruited (12 women and 8 men, aged 18-30 years). Exclusion criteria included: smoking, drinking alcohol, exceeding BMI, oral diseases, pregnancy or lactation, or use of antibiotics during the experiment. Subjects experiencing sample allergies or intolerances during the experiment were also excluded. Ultimately, 18 subjects (10 women and 8 men, aged 18-30 years) completed the experiment. All samples were aliquoted into 30mL tasting cups and coded with three random numbers.

[0057] (3) Sensory evaluator screening and training: Sensory testing was conducted in a standard sensory analysis room. Subjects fasted for at least one hour before the test but were allowed free access to water. First, basic taste sensitivity was assessed using the two-point selection method (2-AFC) according to ISO 5495. The evaluation samples included five basic taste solutions at different concentrations: sodium chloride (15.00 and 3.00 g / L), glucose (50.00 and 10.00 g / L), monosodium glutamate (20.00 and 4.00 g / L), naringin (2.00 and 4.00 mg / L), and citric acid (5.00 and 1.00 g / L). During the test, subjects were required to wear a nasal clip, rinse their mouths with distilled water until no residual taste remained before tasting each sample, and spit out the sample after holding it in their mouth for at least 10 seconds. Only subjects who could correctly identify all differences in taste intensity proceeded to subsequent training. Subsequently, the 18 selected subjects underwent a ranking test. Citric acid (6.00 g / L), naringin (2.00 mg / L), glucose (50.00 g / L), monosodium glutamate (20.00 g / L), and sodium chloride (15.00 g / L) were serially diluted twofold to obtain five concentration gradients. Subjects were required to rank the coded solutions according to perceived intensity. Ultimately, 10 subjects (7 women, 3 men, aged 18–30 years) who performed exceptionally well were selected as members of the formal sensory evaluation panel.

[0058] (4) Evaluation of the flavor intensity of stevia samples: Since the bitterness, grassy taste, and sweetness of stevia culture medium are intertwined, a single standard is difficult to accurately characterize them. Therefore, this invention uses pure stevia solutions of different concentrations as reference standards. The scoring criteria for bitterness and grassy taste are set as follows: 5 points correspond to 7.5 mg / mL, 10 points correspond to 10 mg / mL, and 15 points correspond to 15 mg / mL. Before the formal evaluation, the team members need to be familiar with the flavor characteristics of the above reference solutions. During the evaluation, before tasting each coded sample, the team members need to rinse their mouths with distilled water and wait 30 seconds to eliminate any taste residue. Then, the intensity of bitterness and grassy taste of the samples is scored, with a scoring range of 0 to 15 points. If the flavor intensity of the sample is higher than the 10-point benchmark, the score can be higher than 10 points (maximum 15 points); if the intensity is lower than the 10-point benchmark, the score is reduced accordingly (minimum 0 points).

[0059] (5) Results Analysis: To quantify the flavor improvement effect, this invention uses the flavor decline rate as the core evaluation indicator. The specific calculation formula is as follows: Sensory score decline rate (%) = [(sample score before fermentation - sample score after fermentation) / sample score before fermentation] × 100%. Figure 1It is evident that the undesirable flavor of stevia is significantly improved after fermentation treatment using the shiitake mushroom liquid fermentation process provided by this invention. Specifically, the bitterness decreased by 33.29%, and the grassy taste decreased by 35.98%. This data clearly demonstrates that the composite culture medium based on carrot powder-potato powder-stevia powder and the shiitake mushroom metabolic system can produce an effective synergy, enabling efficient biotransformation of bitter glycosides and herbal volatile substances in stevia, thereby significantly reducing the sensory intensity of its undesirable flavor. This result strongly validates the technical effectiveness of this invention in improving the flavor of stevia, providing crucial sensory data support for its large-scale application as a high-quality natural sweetener in the low-sugar food industry.

[0060] Example 2 Following the compound culture medium preparation and fermentation steps of Example 1, the preparation conditions were as follows: potato flour 4 g / L, carrot flour 10 g / L, stevia powder 2 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L; magnetic stirring speed 1000 rpm, stirring time 30 min; water bath temperature 50℃, water bath time 30 min; sterilization time 15 min. Fermentation conditions: shiitake mushroom seed liquid inoculation amount 20% (volume percentage); culture temperature 20℃; shaker speed 110 rpm; culture time 5 days. The bitterness and grassy taste of the samples before and after fermentation were evaluated according to the sensory evaluation steps in Example 1. Figure 2 It can be seen that after fermentation treatment using the stevia flavor improvement process based on shiitake mushroom liquid fermentation provided by this invention, the bitterness of stevia decreased by 22.63% and the grassy taste decreased by 29.32%.

[0061] Example 3 Following the compound culture medium preparation and fermentation steps of Example 1, the preparation conditions were as follows: potato flour 20 g / L, carrot flour 1 g / L, stevia powder 16 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L; magnetic stirring speed 1500 rpm, stirring time 60 min; water bath temperature 75℃, water bath time 30 min; sterilization time 30 min. Fermentation conditions: 5% (volume percentage) of shiitake mushroom seed liquid inoculation; culture temperature 30℃; shaking speed 90 rpm; culture time 15 days. The bitterness and grassy taste of the samples before and after fermentation were evaluated according to the sensory evaluation steps in Example 1. Figure 3 It can be seen that after fermentation treatment using the stevia flavor improvement process based on shiitake mushroom liquid fermentation provided by the present invention, the bitterness of stevia decreased by 27.96% and the grassy taste decreased by 26.98%.

[0062] Example 4 Following the compound culture medium preparation and fermentation steps of Example 1, the preparation conditions were as follows: potato flour 10 g / L, carrot flour 5 g / L, stevia powder 10 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L; magnetic stirring speed 1500 rpm, stirring time 60 min; water bath temperature 100℃, water bath time 10 min; sterilization time 30 min. Fermentation conditions: 10% (volume percentage) of shiitake mushroom seed liquid inoculation; culture temperature 26℃; shaking speed 110 rpm; culture time 12 days. The bitterness and grassy taste of the samples before and after fermentation were evaluated according to the sensory evaluation steps in Example 1. Figure 4 It can be seen that after fermentation treatment using the stevia flavor improvement process based on shiitake mushroom liquid fermentation provided by the present invention, the bitterness of stevia is reduced by 25.63% and the grassy taste is reduced by 29.25%.

[0063] Example 5 To clarify the effect of fermentation on the sweetness characteristics of stevia, this invention uses sucrose, steviol glycosides (representative of natural sweeteners), and sucralose (representative of artificial sweeteners) as references to conduct equivalent sweetness tests. The following standard solution concentration gradients were set: sucrose solution: 0, 50, 100, 150, 200, 250 mg / mL; steviol glycoside solution: 400, 600, 800, 1000, 1200, 1400 ppm; sucralose solution: 0, 50, 100, 200, 400, 600 ppm. Sensory evaluation was used to determine the standard solution concentrations from which the test sample solution was equivalent in sweetness.

[0064] Sample preparation: Following the preparation steps in Example 1, the preparation conditions were as follows: potato flour 15 g / L, carrot flour 8 g / L, stevia powder 10 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.2 g / L; magnetic stirring speed 1000 r / min; stirring time 30 min; water bath temperature 100℃; water bath time 20 min. The inoculum volume of shiitake mushroom seed liquid was 12% (volume percentage); the culture temperature was 28℃; the shaking speed was 110 r / min; and the culture time was 12 days. After fermentation, the fermentation broth was filtered and pasteurized to obtain the experimental group sample. The control group consisted of unfermented shiitake mushroom culture medium that underwent the same pasteurization treatment.

[0065] Figures 5-7 The results show the equivalent sweetness of the samples before and after fermentation relative to different sweeteners. Figure 5 It can be seen that the sweetness of the unfermented sample (UFer) is equivalent to that of a sucrose solution at 148.3 mg / mL, while the sweetness of the postfermented sample (Fer) is equivalent to that of a sucrose solution at 131.6 mg / mL. There is no significant difference between the two, and the sweetness retention rate is 88.72%. Similarly, Figure 6The results showed that the equivalent sweetness of the samples before and after fermentation relative to steviol glycosides was 888.89 ppm and 794.4 ppm, respectively, with no significant change, and the sweetness retention rate was 89.37%. Figure 7 Further, it was shown that, compared to sucralose, the equivalent sweetness before and after fermentation was 305.56 ppm and 269.44 ppm, respectively, with no significant difference, and the sweetness retention rate was 88.18%.

[0066] In summary, the results show that after fermentation treatment using the stevia flavor enhancement process based on shiitake mushroom liquid fermentation provided by this invention, the equivalent sweetness of the stevia sample relative to sucrose, steviol glycosides, and sucralose did not change significantly, with sweetness retention rates of 88.72%, 89.37%, and 88.18%, respectively. This fully demonstrates that the enhancement process effectively improves the undesirable flavor of stevia while well preserving its core sweetness characteristics, further highlighting the unique advantages of this invention in balancing flavor improvement and sweetness retention.

[0067] The Temporal Dominance of Sensations (TDS) method was employed to comprehensively evaluate the dynamic changes of multiple sensory attributes. TDS aims to dynamically record the changes in the dominant sensory attributes of a sample during the evaluation period, revealing the temporal dominance of these attributes throughout the entire perceptual process, thereby achieving a comprehensive evaluation of the dynamic changes of multiple sensory attributes.

[0068] Figure 8 and Figure 9 The TDS dynamic sensory results are shown for the samples prepared in Example 5 before and after fermentation. In the TDS curve, the significant and random horizontal lines are used to distinguish the dominant sensory attributes. When the sensory attribute curve is above the significant horizontal line, the attribute is considered to be the main dominant sensory feature.

[0069] Depend on Figure 8 It was found that within the sensory cycle of 0-60 seconds, the bitterness, grassy taste, and sweetness of the pre-fermentation sample repeatedly exceeded the significance level, collectively constituting its main sensory characteristics. Specifically, bitterness dominated in three stages: 7-9 seconds, 20-22 seconds, and 33-36 seconds; grassy taste was most dominant during 12-17 seconds, with a maximum dominance rate of 0.65; and sweetness was mainly dominant in 22-32 seconds and 38-43 seconds. In addition, vegetable and astringent tastes were also present, and the overall flavor was less palatable due to unpleasant taste interference.

[0070] Depend on Figure 9As can be seen, after fermentation using the stevia flavor enhancement process based on shiitake mushroom liquid fermentation provided by this invention, the dynamic characteristics of the sample flavor were significantly improved. Although bitterness, grassy taste, and sweetness remained the main sensory attributes, the influence of undesirable flavors was greatly reduced: bitterness only briefly dominated for 26-28 seconds, the dominant period of grassy taste shortened to 9-11 seconds, and the highest dominance rate decreased from 0.65 to 0.53, a decrease of 18.46%. At the same time, the dominant period of sweetness extended to 4-6 seconds in the early stage and 28-41 seconds in the later stage, especially becoming a more persistent and distinct core positive flavor in the later stage. Off-flavors such as vegetable taste and astringency remained at a low level and did not interfere with the overall flavor.

[0071] The TDS analysis above shows that the present invention effectively suppresses the undesirable dominance of bitterness and grassy taste in stevia through fermentation treatment, while retaining the perceived advantage of sweetness. This verifies the significant effect of the improved process in improving the palatability of stevia from a dynamic sensory perspective.

[0072] To elucidate the regulatory effect of shiitake mushroom fermentation on steviol glycoside components, high-performance liquid chromatography (HPLC) was used to qualitatively and quantitatively analyze ribobandi glycoside A (RA), steviol glycoside (STV), ribobandi glycoside F (RF), and ribobandi glycoside C (RC) in the samples before and after fermentation described in Example 5. The HPLC analysis method followed GB 1886.355-2022.

[0073] (1) Preparation of mixed standard solution: Accurately weigh RA, STV, RF and RC standards, and prepare mixed standard solution according to GB 1886.355-2022. The concentration (ug / mL) ratio of the four steviol glycosides is 1:0.2:0.5:0.3 to facilitate the identification of the corresponding chromatographic peaks of each component in the chromatogram.

[0074] (2) HPLC determination conditions Column: C18 (250 mm × 4.6 mm, 5 µm). Mobile phase: Phase A is sodium phosphate buffer; Phase B is acetonitrile. Flow rate: 0.8 mL / min. Injection volume: 10 µL. Column temperature: 40°C. Detection wavelength: 210 nm.

[0075] Gradient elution program

[0076] (3) Measurement steps Under the chromatographic conditions described above, the mixed standard solution, the pre-fermentation sample, and the post-fermentation sample were analyzed sequentially. The chromatographic peaks of each component in the sample were determined by comparing retention times.

[0077] Chromatographic analysis results (see) Figure 10The results showed that the peak areas of RC and STV in the fermented sample decreased significantly, while the peak area of ​​RF increased significantly, and the peak area of ​​RA decreased slightly. Quantification was performed using the external standard method, and the results (see...) Figure 11 The results showed that after fermentation, the retention rate of RA was 89.85% (decreased from 978.6 μg / mL to 879.2 μg / mL); the contents of STV and RC decreased by 18.39% (from 146.8 μg / mL to 119.8 μg / mL) and 13.44% (from 123.328 μg / mL to 106.744 μg / mL), respectively; while the content of RF increased significantly from 17.6 μg / mL to 450.0 μg / mL, an increase of 2414.6%.

[0078] The above results demonstrate that the stevia flavor improvement process based on shiitake mushroom liquid fermentation provided by this invention can directionally regulate steviol glycoside components. Specifically, the glycosidases secreted by the strain can specifically degrade glycosides with undesirable flavors such as STV and RC; while efficiently retaining the core sweet component RA, it can directionally enrich RF. As described in the prior art (CN110381748A), RF has a synergistic enhancing effect on sweetness perception. The above-mentioned changes in chemical components are consistent with the sensory evaluation results (bitterness decreased by 33.29%, and sweetness retention rate exceeded 88%), systematically confirming at the molecular level the synergistic advantages and innovation of the stevia flavor improvement process based on shiitake mushroom liquid fermentation provided by this invention in improving stevia flavor and retaining sweetness characteristics.

Claims

1. A process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms, characterized in that, Includes the following steps: (1) Preparation of fermentation medium: Weigh potato flour, carrot flour and stevia powder, add potassium salt and / or magnesium salt according to fermentation requirements, add to ultrapure water and stir evenly, dissolve in water bath, filter, sterilize at high temperature and high pressure to obtain shiitake mushroom fermentation medium; (2) Inoculation of strain: Inoculate the shiitake mushroom strain seed liquid in the logarithmic growth phase into the shiitake mushroom fermentation medium described in step (1) at an inoculation amount of 5% to 20% by volume; (3) Fermentation culture: The inoculated shiitake mushroom fermentation medium was placed on a shaker and cultured at a constant temperature to obtain stevia fermentation broth samples; (4) Edible treatment: The unstevia fermentation liquid sample was treated to be edible.

2. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (1), the potato flour is weighed at 4~20 g / L, the carrot flour at 1~10 g / L, and the stevia powder at 2~16 g / L.

3. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (1), the potassium salt includes at least one of potassium chloride, potassium dihydrogen phosphate, and potassium sulfate, and the magnesium salt includes at least one of magnesium chloride and magnesium sulfate.

4. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (1), the stirring operation is carried out by magnetic stirring at a speed of 500~1500 r / min and a stirring time of 10~60 min; the water bath dissolution temperature is 50~100℃ and the water bath time is 10~30 min.

5. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (1), according to claim 1, the process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms is characterized in that, in step (1), the high temperature and high pressure sterilization time is 15~40min.

6. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (1), the filtration medium includes at least one of coarse cotton cloth, coffee filter paper, and 0.8-micron filter.

7. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (2), the shiitake mushroom is a strain of Lentinula edodes, including but not limited to at least one of the following: shiitake mushroom L9608, shiitake mushroom L241-4, shiitake mushroom B08, shiitake mushroom CPCC 400023, shiitake mushroom ACCC 52357, shiitake mushroom Anxiang No. 2 L18-1, shiitake mushroom L135-1, shiitake mushroom 808 and shiitake mushroom L9015.

8. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (2), the shiitake mushroom is shiitake mushroom ACCC 52357.

9. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (3), the shaking speed is 90~150 r / min, and the constant temperature culture is carried out at 20~30℃ for 5~15 days.

10. The process for improving the undesirable flavor of stevia based on liquid fermentation of shiitake mushrooms according to claim 1, characterized in that, In step (4), the stevia fermentation broth sample is processed using techniques including pasteurization and ultra-high temperature instantaneous sterilization.

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