Rosa roxburghii fermentation process based on non-saccharomyces cerevisiae and application technology

Abstract

The invention relates to the technical field of food fermentation, and particularly discloses a rosa roxburghii fermentation process based on non-saccharomyces cerevisiae and an application technology. According to the present invention, the non-saccharomyces cerevisiae YC2 is screened from the yeast for making hard liquor, and the non-saccharomyces cerevisiae YC2 is identified as the saccharomycopsis fibuligera, and has characteristics of good probiotic property, good tolerance and good fermentation adaptability; fermentation process parameters are optimized through a response surface analysis method, and the optimal conditions are determined as follows: the fermentation time is 4 days, the fermentation temperature is 33 DEG C, the material-liquid ratio is 60%, and the inoculum size is 4%. The strain is used for fermenting roxburgh rose juice, so that the sensory quality, antioxidant activity and bioactive substance content of the product can be remarkably improved, and the strain has the advantages of unique flavor, strong functionality, high fermentation efficiency and the like, and is suitable for industrial production.

Description

Technical Field

[0001] This invention relates to the field of food fermentation technology, specifically a prickly pear fermentation process and application technology based on non-Saccharomyces cerevisiae. Background Technology

[0002] Prickly pear is a natural fruit rich in vitamins, bioactive substances, and antioxidants, possessing high nutritional and health value. Currently, prickly pear products are mostly available as juice or blended beverages. Juice has a poor taste, while blended beverages contain many food additives, which does not align with health-conscious consumption trends. Fermentation technology can effectively improve the flavor of prickly pear juice, enhance the bioavailability of its nutrients, and endow the product with prebiotic functions.

[0003] Traditional prickly pear fermentation relies heavily on brewer's yeast or natural fermentation microorganisms, resulting in issues such as limited flavor, unstable fermentation efficiency, and insufficient functionality. Non-brewing yeasts, however, possess unique advantages in juice fermentation due to their ability to produce abundant aroma compounds and extracellular enzymes. Nevertheless, current technologies lack dedicated non-brewing yeast strains specifically screened for the characteristics of prickly pear, and fermentation processes often employ single-factor optimization, failing to fully consider the interactions between various factors, thus hindering further improvements in product quality.

[0004] Therefore, screening non-sacchariculture yeast strains suitable for prickly pear fermentation and establishing a scientific and efficient fermentation process are of great significance for developing high-quality prickly pear fermented products. Summary of the Invention

[0005] This invention belongs to the field of food fermentation technology, specifically relating to a method for screening non-saccharifying yeasts, a prickly pear juice fermentation process and its application, and particularly to screening non-saccharifying yeasts from koji (a type of starter culture) and optimizing their process parameters in prickly pear juice fermentation.

[0006] To achieve the above objectives, the present invention provides the following technical solution:

[0007] A screening method for non-Saccharomyces cerevisiae. This method overcomes the limitations of relying on commercial strains, starting from the natural carrier of Daqu (a type of starter culture) in the brewing environment of Maotai-flavor liquor, and obtains a specific strain suitable for prickly pear juice fermentation through a multi-round screening system including the following steps:

[0008] 1) Isolation and purification: Single colonies were isolated from Daqu using YPD solid medium;

[0009] 2) Morphological screening: Yeasts with multi-terminal budding reproduction characteristics are screened by microscopic examination;

[0010] 3) Physiological and biochemical rescreening: Using WL differential medium and lysine-deficient medium, non-Saccharomyces cerevisiae and Saccharomyces cerevisiae were initially distinguished;

[0011] 4) Molecular biological identification: 26S rDNA sequence analysis was performed on suspected strains and compared with the NCBI database to determine their species;

[0012] 5) Evaluation of probiotic characteristics: The self-agglutination ability, hydrophobicity and in vitro antioxidant capacity (such as DPPH free radical scavenging rate) of the identified strains were measured to screen out strains with good fermentation adaptability and potential probiotic functions.

[0013] Using the above methods, a high-performing non-Saccharomycopsis strain was finally screened and named YC2, which was identified as Saccharomycopsis fibuligera.

[0014] A process for fermenting prickly pear juice using the non-Saccharomyces cerevisiae YC2 obtained from the above screening. The core of this process lies in the scientific optimization of key fermentation parameters using response surface methodology, specifically including:

[0015] 1) The sensory quality score of fermented prickly pear juice (comprehensive taste, aroma, mouthfeel, texture and color) was used as the response value;

[0016] 2) Fermentation time, fermentation temperature, material-to-liquid ratio (percentage of prickly pear pulp), and inoculum size were selected as key influencing factors;

[0017] 3) Using Box-Behnken experimental design, a quadratic polynomial mathematical model was established between each factor and the response value to analyze the interaction between factors;

[0018] 4) Through model solving and verification experiments, the optimal fermentation process parameters were determined to be: fermentation time 4 days, fermentation temperature 33℃, material-to-liquid ratio 60%, and inoculum size 4%.

[0019] This optimized process significantly improves fermentation efficiency and the stability and predictability of product sensory quality.

[0020] The application of fermentation technology in the preparation of prickly pear fermented products, and the prickly pear fermented products obtained by this method. This product not only retains the nutrients of the prickly pear raw material, but also significantly improves the content or bioavailability of bioactive substances such as total phenols and vitamin C through microbial fermentation, giving the product stronger antioxidant activity and a unique flavor profile.

[0021] Compared with the prior art, the beneficial effects of the present invention are:

[0022] Innovation and adaptability of strains: YC2, a non-sacchariculture yeast strain with good adaptability to prickly pear substrate, was successfully screened from the traditional fermentation environment, which solved the problems that commercial general yeast strains may have in prickly pear fermentation, such as single flavor contribution and unstable fermentation efficiency.

[0023] The process optimization is highly scientific: it abandons the traditional single-factor empirical optimization and adopts the response surface methodology to systematically quantify the interaction effects between multiple factors. The resulting combination of process parameters is more accurate and reliable, which is conducive to quality control in industrial production.

[0024] Product Function and Quality Improvement: The YC2 strain itself possesses excellent probiotic properties (such as high hydrophobicity, self-aggregation, and antioxidant capacity), and its fermentation process can effectively enrich or transform the active ingredients in prickly pear. Experiments have confirmed that the fermented prickly pear juice obtained is superior to products using commercial control strains in key functional indicators such as DPPH and ABTS free radical scavenging rates and total phenolic content.

[0025] The technology boasts high replicability and broad application prospects: the provided strain screening methods and fermentation process optimization paths are clear and well-defined, exhibiting excellent repeatability and scalability. The resulting products possess nutritional, delicious, and health-promoting attributes, providing a new technical solution for the deep processing of prickly pear and the development of high-value-added fermented beverages. Attached Figure Description

[0026] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0027] Figure 1 Microscopic images of non-Saccharomyces cerevisiae YC2 (showing spherical or ellipsoidal cell morphology and multi-terminal budding reproduction);

[0028] Figure 2 Results of acetic acid tolerance assay for non-Saccharomyces cerevisiae YC2;

[0029] Figure 3 Results of bile salt tolerance test of non-Saccharomyces cerevisiae YC2; Figure 4 Results of hydrophobicity determination of non-Saccharomyces cerevisiae YC2;

[0030] Figure 5 Results of the self-agglutination ability determination of non-Saccharomyces cerevisiae YC2;

[0031] Figure 6 Comparison of autoagglutination rates between non-Saccharomyces cerevisiae YC2 and AQ strains;

[0032] Figure 7 Comparison of changes in soluble solids content between non-Saccharomyces cerevisiae YC2 and AQ strains;

[0033] Figure 8Comparison of reducing sugar content changes between non-Saccharomyces cerevisiae YC2 and AQ strains;

[0034] Figure 9 Comparison of pH changes between non-Saccharomyces cerevisiae YC2 and AQ strains;

[0035] Figure 10 Comparison of DPPH free radical scavenging rates between non-Saccharomyces cerevisiae YC2 and KB strains, and vitamin C;

[0036] Figure 11 Results of changes in DPPH free radical scavenging rate in prickly pear juice fermented with non-Saccharomyces cerevisiae YC2;

[0037] Figure 12 Results of changes in ABTS free radical scavenging rate in prickly pear juice fermented with non-Saccharomyces cerevisiae YC2. Detailed Implementation

[0038] In the description of this invention, it should be understood that the terms "center", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", etc., indicate the orientation or positional relationship based on the orientation or positional relationship shown in the accompanying drawings. They are only for the convenience of describing this invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific orientation, or be constructed and operated in a specific orientation. Therefore, they should not be construed as limitations on this invention.

[0039] In the description of this invention, it should be noted that, unless otherwise explicitly specified and limited, the terms "installation," "connection," and "configuration" should be interpreted broadly. For example, they can refer to a fixed connection or configuration, a detachable connection or configuration, or an integral connection or configuration. Those skilled in the art can understand the specific meaning of the above terms in this invention based on the specific circumstances.

[0040] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0041] Example 1: Screening and molecular identification of non-Saccharomyces cerevisiae YC2

[0042] Strain isolation: Weigh 10g of Daqu sample, add 90mL of sterile physiological saline, shake well to prepare a suspension, and perform serial dilutions. Take 100μL of the suspension at an appropriate dilution and spread it on YPD solid medium (formulation: yeast extract 10g / L, peptone 20g / L, glucose 20g / L, agar 30g / L, pH natural) plates, and incubate upside down at 28℃ for 48-72h. Select single colonies with obvious morphological differences for repeated streak purification.

[0043] Morphological screening: The purified strains were prepared into water slides and observed under an optical microscope (1000x oil immersion). Strains with spherical or ellipsoidal cell shapes and that reproduce via multi-terminal budding were selected (see...). Figure 1 ).

[0044] Physiological and biochemical rescreening: The initially screened strains were inoculated onto WL differential medium and lysine auxotrophic medium plates, respectively, and incubated at 28℃ for 3-5 days. The colony color, morphology, and growth on lysine medium were observed to preliminarily distinguish between non-Saccharomyces cerevisiae and Saccharomyces cerevisiae.

[0045] Molecular biological identification: Genomic DNA was extracted from suspected non-Saccharomycopsis strains, and the 26S rDNA D1 / D2 region was amplified using universal fungal primers NL1 / NL4. The PCR products were sequenced, and the sequences were submitted to the NCBI database for BLASTn homology comparison. The results showed that one strain (number YC2) had a sequence similarity of 99% with *Saccharomycopsis fibuligera*, and therefore it was identified as *Saccharomycopsis fibuligera*.

[0046] Strain preservation: The identified YC2 strain was inoculated onto YPD slant medium, cultured at 28°C until mature, and then stored in a 4°C freezer for a short period of time, or frozen at -80°C using 20% ​​glycerol tubes.

[0047] Example 2: Tolerance evaluation of strain YC2

[0048] Acetic acid tolerance: Activated YC2 seed culture was inoculated at a rate of 2% (v / v) into YPD liquid medium containing 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, and 1.2% (v / v) acetic acid, respectively. After incubation at 36℃ and 210 r / min for 36 h with shaking, the absorbance (OD600) of the fermentation broth was measured at 600 nm. Results ( Figure 2 The results showed that YC2 grew well when the acetic acid concentration was ≤0.6%; when the concentration increased to 1.2%, the growth was basically inhibited (OD600=0.261).

[0049] Temperature tolerance: YC2 seed culture was inoculated into YPD liquid medium at a 2% inoculum and incubated at 26℃ and 37℃ for 48 h, respectively. OD600 was then measured. Results ( Figure 3 The results showed that YC2 could still maintain active growth at human body temperature (37℃) with a large cell density, indicating that it has a wide range of temperature adaptability.

[0050] Bile salt tolerance: YC2 bacterial suspension was serially diluted, and 100 μL was spread onto YPD solid agar plates containing 0%, 0.03%, 0.1%, 0.2%, and 0.3% (w / v) porcine bile salts, respectively. After incubation at 37℃ for 24 h, colony growth was observed and counted. Calculations showed that even at a high bile salt concentration of 0.3%, YC2 maintained a high survival rate. Figure 4 This demonstrates good potential for tolerance to the gastrointestinal fluid environment.

[0051] Example 3: Determination of the probiotic characteristics of strain YC2

[0052] Autoagglutination ability: YC2 cells were cultured to the stationary phase, collected by centrifugation, washed twice with PBS buffer, and resuspended to OD600 = 1.0. 4 mL of the bacterial suspension was placed in a test tube and incubated at 37°C for 2 hours. The supernatant (200 μL) was carefully aspirated to determine OD600. The OD600 value at 0 hours was A0, and the OD600 value at 2 hours was At. The autoagglutination rate (%) was calculated as (1 - At / A0) × 100%. A commercial probiotic, *Saccharomyces boulardii*, was used as a control. Results ( Figure 6 The results showed that the self-aggregation rate of YC2 increased over time, reaching a high level after 2 hours, indicating that it has good self-aggregation ability, which helps it to colonize in the intestine.

[0053] Hydrophobicity: Prepare 3 mL of a bacterial suspension with OD600 = 1.0 using the same method as above, add 1 mL of n-hexadecane, vortex vigorously for 2 min to mix thoroughly, let stand at 37℃ for 30 min to separate the layers, and then measure the OD600 of the lower aqueous phase. Hydrophobicity (%) = (1 - A / A0) × 100%, where A0 is the OD600 of the bacterial suspension before mixing, and A is the OD600 of the aqueous phase after separation. Results ( Figure 5 The results showed that YC2 has a hydrophobicity of over 80%, classifying it as a highly hydrophobic strain, which suggests that it can adhere well to intestinal epithelial cells.

[0054] In vitro antioxidant capacity: The supernatant of YC2 fermentation broth was mixed with an equal volume of 0.1 mmol / L DPPH-ethanol solution and reacted in the dark for 30 min. The absorbance was then measured at 517 nm. Ethanol was used as a blank instead of the supernatant, and distilled water was used as a background control instead of the DPPH solution. DPPH free radical scavenging rate (%) = [1 - (Asample - Abackground) / Ablank] × 100%. The experiment showed that the YC2 fermentation supernatant had a significant DPPH free radical scavenging capacity.

[0055] Example 4: Optimization of Prickly Pear Juice Fermentation Process Based on Response Surface Methodology

[0056] Experimental Design: YC2, obtained through screening, was used as the fermentation strain. The ratio of prickly pear pulp to water (solid-liquid ratio), fermentation temperature, fermentation time, and inoculum size were the four key independent variables. Based on the single-factor preliminary experiment, a Box-Behnken design (BBD) was used to conduct a four-factor, three-level response surface methodology experiment. The overall sensory quality score of the final fermented product (out of 100 points, evaluated by a trained review panel based on taste (20 points), aroma (20 points), mouthfeel (30 points), texture (20 points), and color (10 points)) was used as the response value Y.

[0057] Model Establishment and Optimization: Multiple regression fitting was performed on the experimental data using Design-Expert software to obtain a quadratic polynomial model equation relating sensory score Y to the coded values ​​of each factor (X1: fermentation time, X2: fermentation temperature, X3: solid-liquid ratio, X4: inoculum size). Analysis of variance showed that the model was significant (p<0.01), with no significant lack-of-fit terms, indicating a good model fit. Analysis of response surface plots (such as...) further validated the model. Figure 7-10 Example) and contour plots to examine the interactions between various factors.

[0058] Optimal process determination: The optimal process parameter combination was determined by solving the model using software: fermentation time 4 days, fermentation temperature 33℃, material-to-liquid ratio 60% (i.e., prickly pear pulp content), and inoculum size 4%. Validation experiments were conducted under these conditions, and the actual sensory scores showed good agreement with the model predictions, confirming the reliability of the model.

[0059] Example 5: Performance Comparison of YC2 Fermented Prickly Pear Juice and Commercial Yeast Fermentation Products

[0060] Sample preparation: The same batch of prickly pear pulp was fermented using the optimal process of this invention (YC2 strain) and using the same fermentation parameters but with commercially available Angel Yeast aroma-producing active dry yeast (control).

[0061] Antioxidant activity comparison: The DPPH and ABTS free radical scavenging rates of the two fermentation juices were determined. Results (e.g.) Figure 11 ,12 As shown in the figure, the free radical scavenging rate of the YC2 fermentation group was consistently higher than that of the commercial yeast control group throughout the fermentation process, and the difference was significant after the fermentation was completed (p<0.05).

[0062] Comparison of bioactive substance content: The contents of vitamin C, total phenols, total flavonoids, and soluble protein in the two fermentation products were determined before and after fermentation. The results (summarized in Table 1) showed that after YC2 fermentation, the contents of total phenols and vitamin C in the prickly pear juice increased significantly compared with those before fermentation (from 119.44 mg / 100 mL to 168.81 mg / 100 mL, and from 402.23 mg / 100 mL to 445.10 mg / 100 mL, respectively), while the contents of total flavonoids and protein decreased. Compared with commercial yeast fermentation products, YC2 fermentation products showed better retention or enhancement of total phenols and vitamin C.

[0063] Preliminary analysis of flavor compounds: Preliminary analysis using headspace solid-phase microextraction combined with gas chromatography-mass spectrometry (HS-SPME-GC-MS) revealed that the types and relative contents of flavor compounds such as esters and alcohols in YC2 fermented prickly pear juice differed from those in commercial yeast fermentation products, which may contribute to its richer aroma profile.

[0064] The non-Saccharomyces cerevisiae YC2 screening method provided by this invention is stable and reliable, and can be used to selectively screen functional strains from various traditional fermented foods. The established optimized fermentation process parameters for prickly pear juice are well-defined and reproducible, and can be directly used to guide industrial production. Prickly pear fermented beverages produced using this technology not only have excellent sensory quality but are also rich in antioxidant active ingredients, aligning with current health beverage consumption trends and possessing good market conversion potential. Furthermore, this strain and process can also provide a reference for the fermentation improvement of other specialty fruit juices.

[0065] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above, and that the invention can be implemented in other specific forms without departing from its spirit or essential characteristics. Therefore, the embodiments should be considered in all respects as exemplary and non-limiting, and the scope of the invention is defined by the appended claims rather than the foregoing description. Thus, all variations falling within the meaning and scope of equivalents of the claims are intended to be included within the present invention. No reference numerals in the claims should be construed as limiting the scope of the claims.

[0066] Furthermore, it should be understood that although this specification describes embodiments, not every embodiment contains only one independent technical solution. This narrative style is merely for clarity. Those skilled in the art should consider the specification as a whole, and the technical solutions in each embodiment can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

Claims

1. A non-Saccharomyces-based roxburghii fermentation process and application technology, comprising the following steps: Using Daqu as raw material, separating and purifying through YPD solid medium to obtain single colonies; Screening strains with multiple end-budding propagation through microscope examination; Re-screening through WL agar medium and preliminary classification through lysine medium to obtain suspected non-Saccharomyces strains; Identifying through 26S rDNA sequence to confirm that it is non-Saccharomyces strain YC2, and the sequence similarity with existing non-Saccharomyces sequence in NCBI is 99%.

2. The screening method according to claim 1, characterized in that, The composition of the YPD solid medium is: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, and agar 30 g / L.

3. The screening method according to claim 1, characterized by, The non-Saccharomyces strain YC2 has the following probiotic properties: self-aggregation ability, hydrophobicity, and DPPH free radical scavenging ability.

4. A roxburghii juice fermentation process characterized in that, The non-Saccharomyces strain YC2 obtained by screening according to any one of claims 1-3 is used as the fermentation strain, and the following fermentation parameters are obtained through response surface analysis: fermentation time 4 days, fermentation temperature 33℃, solid-liquid ratio 60%, and inoculum size 4%.

5. The Malus sieversii juice fermentation process according to claim 4, characterized in that, The response surface analysis uses Box-Behnken design, takes fermentation time, fermentation temperature, solid-liquid ratio, and inoculum size as influencing factors, takes sensory quality score as response value, establishes a quadratic polynomial mathematical model for optimization.

6. A method for preparing a roxburghy melon fermented product, characterized by, Comprising the following steps: Using the fermentation process according to claim 4 or 5 to ferment roxburghii juice; After fermentation, sterilization, filtration, and filling are performed to obtain roxburghii fermented juice product.

7. The preparation method according to claim 6, characterized in that, The total phenol and vitamin C content in the roxburghii fermented juice product is significantly increased compared to before fermentation, and the flavonoid and protein content is decreased.

8. Use of a non-Saccharomyces cerevisiae yeast YC2 in the preparation of a fermented product of Physalis pruinosa, characterized in that, The strain is used to improve the sensory quality, antioxidant activity, and bioactive substance content of roxburghii juice.

9. Use according to claim 8, characterized in that, The non-Saccharomyces strain YC2 is Saccharomycopsis fibuligera.

10. A roxburgh raspberry fermented product, characterized by, Prepared by the method of claim 6 or 7, with enhanced antioxidant activity, rich flavor components, and probiotic function.

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