Preparation method of sugar-free lily original syrup beverage fermented by probiotics

Abstract

The present application relates to the technical field of beverage processing, and particularly relates to a preparation method of a probiotic fermented sugar-free lily original syrup beverage. The method comprises the following steps: obtaining lily slurry by beating lily bulb raw materials after treatment; performing three-stage gradient enzymolysis on the lily slurry to convert starch into fermentable sugar, and controlling the reducing sugar content at 4.0-6.0 g / 100 mL; after cooling and adjusting the pH of the enzymolysis liquid, inoculating Bacillus amyloliquefaciens for pre-fermentation, inoculating Lactobacillus plantarum and Bifidobacterium breve complex inoculum for main fermentation until the pH is reduced to 3.8-4.5; and finally performing high-pressure micro-jet homogenization and sterilization treatment on the fermentation liquid. The present application can realize the preparation of a probiotic fermented sugar-free lily original syrup beverage with low residual sugar, high viable count and no stratified precipitation within the shelf life without additional sugar source and chemical thickening agent.

Description

Technical Field

[0001] This invention relates to the field of beverage processing technology, and more specifically, to a method for preparing a probiotic fermented sugar-free lily pulp beverage and the probiotic fermented sugar-free lily pulp beverage obtained by the method. Background Technology

[0002] Lily, a traditional plant used in both medicine and food, is rich in active ingredients such as polysaccharides, saponins, and amino acids, making it a promising candidate for development in the health food and beverage industry. With increasing consumer focus on healthy eating, fermented beverages made from lily are becoming a market hotspot. Existing lily fermentation processes typically include raw material washing and pulping, enzymatic hydrolysis, inoculation and fermentation, and post-processing sterilization. In conventional techniques, to provide the carbon source needed for probiotic growth, sugary additives such as sucrose, glucose, or milk powder are often added to the lily pulp. Simultaneously, to address instability caused by protein denaturation and fiber precipitation during fermentation, chemical thickeners or stabilizers such as xanthan gum and sodium carboxymethyl cellulose are usually added. Furthermore, existing processes often employ a single-strain or mixed-strain inoculation method for fermentation, relying on empirical control over a fixed time frame to complete the entire fermentation process, and finally using conventional homogenization equipment to refine the product.

[0003] However, in existing technologies, due to the high starch content of lily raw materials, which are difficult for probiotics to utilize directly, fermentation is insufficient without the addition of exogenous sugars, while the addition of exogenous sugars makes it impossible for the product to meet the sugar-free health requirements. At the same time, relying on chemical additives to maintain stability does not conform to the trend of clean labeling, and the extensive fermentation control method is difficult to cope with batch differences in raw materials, resulting in fluctuations in residual sugar content, live bacteria count, flavor consistency and shelf-life stability of the final product, making it difficult to simultaneously achieve the comprehensive quality requirements of sugar-free, high live bacteria count and long-term physical stability. Summary of the Invention

[0004] In view of the problems existing in the prior art, the purpose of this invention is to provide a method for preparing a probiotic fermented sugar-free lily pulp beverage.

[0005] In a first aspect, the present invention provides a method for preparing a probiotic fermented sugar-free lily puree beverage, comprising the following steps: S1. Raw material processing: The lily bulb raw material is washed, sliced, and treated with at least one of the following methods: soaking in citric acid, blanching for color protection, and vacuum color protection. Then, 1 to 3 times the weight of purified water is added and pulped to obtain lily pulp. S2. Gradient enzymatic hydrolysis: The lily slurry is subjected to a three-stage gradient enzymatic hydrolysis to convert the starch in it into fermentable sugars. The hydrolysis endpoint is achieved when the reducing sugar content is 4.0~6.0g / 100mL. S3. Segmented Fermentation: Cool the lily enzymatic hydrolysate obtained after enzymatic hydrolysis to 35-40℃ and adjust the pH to 6.0-7.0. The segmented fermentation includes: S31, Pre-fermentation section: Bacillus amyloliquefaciens is inoculated into the lily enzymatic hydrolysate, and fermentation is carried out at 35~40℃; S32. Main fermentation stage: After the pre-fermentation is completed, a compound bacterial agent containing Lactobacillus plantarum and Bifidobacterium breve is inoculated into the system, and the main fermentation is carried out at 35~40℃ until the pH of the fermentation broth drops to 3.8~4.5. S4. Post-processing: After the main fermentation is completed, the fermentation liquid is subjected to high-pressure micro-jet homogenization at a pressure of 60~150MPa, and then sterilized to obtain the probiotic fermented sugar-free lily puree beverage.

[0006] Preferably, the three-stage gradient enzymatic hydrolysis includes: First stage of enzymatic hydrolysis: Add α-amylase and carry out enzymatic hydrolysis at 60~75℃; The second stage of enzymatic hydrolysis: add saccharifying enzyme and carry out enzymatic hydrolysis at 55~65℃ and pH 4.0~5.5; The third stage of enzymatic hydrolysis: add debranching enzyme and carry out enzymatic hydrolysis at 55~65℃.

[0007] The α-amylase is selected from at least one of mesophilic α-amylase, thermostable α-amylase, and bacterial α-amylase; the saccharifying enzyme is selected from at least one of glucoamylase, β-amylase, and fungal saccharifying enzyme; and the debranching enzyme is selected from at least one of pullulanase and isoamylase.

[0008] In the gradient enzymatic hydrolysis, the reducing sugar content at the end of the hydrolysis is 4.5~5.5g / 100mL, and the DE value of the lily hydrolysate is ≥80%.

[0009] In the segmented fermentation process, the fermentation time of the pre-fermentation stage is 4 to 10 hours, and the pH is maintained at ≥5.0 during the fermentation process; the fermentation time of the main fermentation stage is 8 to 30 hours.

[0010] The criteria for determining the end of the main fermentation stage are that all three of the following conditions are met simultaneously: the pH of the fermentation broth is 4.0~4.5, and the total number of viable bacteria is ≥1×10⁻⁶. 9 CFU / mL, lactic acid content ≥4.0g / L.

[0011] The gradient enzymatic hydrolysis in step S2 further includes an adaptive determination of the hydrolysis endpoint based on detection data analysis. The determination method includes: real-time monitoring of the reducing sugar content RS_t and DE value D_t during the hydrolysis process; constructing a discrete sliding window of length N and calculating the weighted moving average Z_t of D_t within the window; dynamically generating an adaptive DE value determination threshold D_th based on the fluctuation of RS_t within the sliding window; and determining the hydrolysis endpoint when Z_t exceeds D_th M times consecutively and RS_t is within the range of 4.5~5.5 g / 100 mL.

[0012] The main fermentation in step S3 further includes determining the fermentation endpoint based on multi-parameter fusion and time-series debouncing. The determination method includes: real-time monitoring of the pH value, lactic acid content L_t, and logarithmic number of viable bacteria C_t of the fermentation broth during the main fermentation process; constructing a comprehensive fermentation index F_t=w1A+w2B+w3*C, where A=4.5-pH, B=L_t / L_ref (L_ref is the preset target lactic acid content), C=C_t / C_ref (C_ref is the preset target logarithmic number of viable bacteria), w1, w2, and w3 are weighting coefficients and their sum is 1; setting an asymmetric hysteresis interval [F_low, F_high], where fermentation is determined to end when F_t is continuously lower than F_low multiple times, and continuous fermentation is determined when F_t is continuously higher than F_high multiple times.

[0013] The high-pressure microfluidic homogenization in step S4 further includes adaptive control of the number of homogenization cycles based on particle size feedback. The control method includes: after each homogenization process, detecting the average particle size d_n of the fermentation broth; using the target particle size d_tar as a set value, calculating the current particle size deviation e_n = d_n - d_tar, and accumulating the deviation integral Sn; based on the discrete proportional-integral control law, predicting the particle size d_{n+1}pred after the next homogenization according to d_{n+1}pred = d_n - K_pe_n - K_iS_n (where K_p is a preset proportional coefficient and K_i is a preset integral coefficient); stopping homogenization when d_{n+1}pred ≤ d_tar, otherwise continuing the next homogenization cycle.

[0014] In step S4, the high-pressure micro-jet homogenization adopts a homogenized nozzle with a built-in self-resonant enhanced cavitation structure. The nozzle outlet front end is provided with a Helmholtz resonant cavity. The inner surface of the resonant cavity is processed with microstructures such as spiral grooves and convex point arrays to guide the fluid to generate swirling flow and excite cavitation effect.

[0015] The beneficial effects of this invention are as follows: This method obtains lily slurry by washing, slicing, color protection, and pulping lily bulb raw materials. Then, a three-stage gradient enzymatic hydrolysis is performed on the lily slurry to accurately convert endogenous starch into fermentable sugars with a reducing sugar content controlled at 4.0~6.0g / 100mL. On this basis, the enzymatic hydrolysate is cooled and the pH is adjusted. It is then inoculated with Bacillus amyloliquefaciens for pre-fermentation. After the pre-fermentation is completed, a compound inoculum composed of Lactobacillus plantarum and Bifidobacterium breve is inoculated for main fermentation until the pH drops to 3.8~4.5. Subsequently, the main fermentation liquid is subjected to high-pressure micro-jet homogenization treatment at 60~150MPa and sterilized. By employing a three-stage gradient enzymatic hydrolysis strategy, the synergistic effects of different enzyme systems under specific temperature and pH conditions are utilized to efficiently and controllably hydrolyze lily endogenous starch into fermentable sugars of appropriate concentrations. This avoids the introduction of external sugar sources and ensures sufficient and precise carbon sources for subsequent fermentation. Based on a segmented fermentation mechanism, *Bacillus amyloliquefaciens* is used to degrade macromolecules and optimize the substrate environment during the pre-fermentation stage. Then, *Lactobacillus plantarum* and *Bifidobacterium breve* are used for synergistic metabolic acid production and proliferation during the main fermentation stage, resulting in enriched flavor compounds and the accumulation of high viable cell counts. The strong shearing and cavitation effects generated by high-pressure microfluidic homogenization promote the micronization of lily dietary fiber and the formation of a stable three-dimensional suspension network, thus replacing the use of chemical thickeners. This effectively solves the problems of high sugar content due to reliance on external sugar sources, reliance on chemical additives to maintain stability, and batch quality fluctuations caused by rough fermentation process control in traditional processes. This results in the preparation of sugar-free, clean-label, high-viable-cell-count, and shelf-stable lily pulp beverages, improving the food safety and functional value of the product. In summary, this application constructs a complete technical closed loop from endogenous sugar conversion and microbial synergistic metabolism to the formation of a physically self-stabilizing system by organically coupling four core steps: raw material processing, gradient enzymatic hydrolysis, segmented fermentation, and high-pressure microfluidic homogenization. This not only significantly improves the bioavailability of lily raw materials and the consistency of product quality, but also provides a systematic solution for the green manufacturing of functional plant protein beverages. Detailed Implementation

[0016] To enable those skilled in the art to better understand the present application, the technical solutions in the embodiments of the present application will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present application, and not all embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present application.

[0017] The present application will now be described in further detail with reference to embodiments. It is to be understood that the specific embodiments described herein are merely illustrative of the present application and are not intended to limit the scope of the application.

[0018] The technical solution of the present invention is illustrated below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not preclude the existence of other method steps before or after the combined steps, or the insertion of other method steps between these explicitly mentioned steps. It should also be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Furthermore, unless otherwise stated, the numbering of each method step is merely a convenient tool for identifying each method step, and not for limiting the order of the method steps or restricting the scope of the present invention. Changes or adjustments to their relative relationships, without substantially altering the technical content, should also be considered within the scope of the present invention.

[0019] To better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. While exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the invention to those skilled in the art.

[0020] The present invention will be further described below with reference to the following embodiments.

[0021] Unless otherwise specified, all materials, reagents and instruments used in the embodiments of this invention can be obtained through commercial channels.

[0022] 1. Materials and Reagents Key ingredients: Fresh lily bulbs (purchased from an agricultural cooperative in Lanzhou, Gansu Province, variety: Lanzhou lily); *Bacillus amyloliquefaciens* (commercially available); *Lactobacillus plantarum* (commercially available); *Bifidobacterium breve* (commercially available); α-amylase (mesothermal type, activity 20000 U / g), glucoamylase (glucosidase, activity 100000 U / g), and debranching enzyme (pullulanase, activity 8000 U / g), all purchased from a biotechnology company; purified water conforming to GB 17323 standard.

[0023] 2. Instruments and Equipment Colloid mill (model JM-L80, manufactured by a machinery factory in Wenzhou); enzymatic hydrolysis tank (with temperature control and stirring system, 50L capacity); fermentation tank (with online pH monitoring and automatic feeding system, 50L capacity); high-pressure microfluidic homogenizer (model AH-1500, manufactured by a nanotechnology company in Shanghai); fully automated biochemical analyzer (for reducing sugar and lactic acid determination); particle size analyzer (for particle size determination); constant temperature incubator; clean bench.

[0024] 3. Characterization and Testing Methods Determination of reducing sugar content: The DNS colorimetric method was used, in accordance with GB / T 5009.7. DE value determination: Fehling's reagent titration method combined with reducing sugar content calculation; Total viable count: Plate count method was used. Lactobacillus plantarum was cultured anaerobically on MRS agar medium, Bifidobacterium breve was cultured anaerobically on TPY agar medium, and Bacillus amyloliquefaciens was cultured aerobically on LB agar medium. Lactic acid content determination: High performance liquid chromatography (HPLC) was used, with a C18 column and potassium dihydrogen phosphate solution as the mobile phase. Particle size distribution was determined using dynamic light scattering (DLS). Stability test: The sample was placed at a constant temperature of 25℃ for 6 months to observe whether there was any layering or precipitation, and the difference in solid content between the upper layer and the lower layer was measured.

[0025] Example 1: This embodiment provides a method for preparing a probiotic fermented sugar-free lily puree beverage under optimal process parameters, verifying the core feasibility and best technical effect of the present invention.

[0026] The steps are as follows: S1. Raw material processing: Select fresh lily bulbs, peel and wash them, then cut them into 3mm thin slices. Soak them in a 0.1% citric acid solution for color protection for 20 minutes. After draining, add twice the amount of purified water and grind them in a colloid mill at 10,000 rpm for 2 minutes to obtain lily pulp with a fineness of no more than 100 microns.

[0027] S2. Gradient Enzymatic Hydrolysis: The lily slurry was heated to 68℃, and 15 U / g starch-containing mesophilic α-amylase was added. The mixture was stirred and hydrolyzed for 45 minutes to complete the first stage of liquefaction. Subsequently, the pH of the slurry was adjusted to 4.8, and the temperature was lowered to 60℃. 150 U / g starch-containing glucoamylase (a combination of glucoamylase and β-amylase) was added, and hydrolysis was carried out for 90 minutes to complete the second stage of saccharification. The temperature was maintained at 60℃, and 8 U / g starch-containing pullulanase was added, hydrolysis was carried out for 40 minutes to complete the third stage of debranching. The hydrolysis process was monitored in real time. When the reducing sugar content reached 5.0 g / 100 mL and the DE value was 88%, the temperature was raised to 90℃ for 10 minutes to inactivate the enzyme, yielding the lily hydrolysate.

[0028] S3. Segmented fermentation: Cool the lily enzymatic hydrolysate to 37°C and adjust the pH to 6.5.

[0029] S31, Pre-fermentation section: Inoculate at a rate of 5×10 6 CFU / mL of Bacillus amyloliquefaciens was inoculated and fermented at 37°C for 6 hours, during which sterile ammonia was added dropwise to maintain pH ≥ 5.0. After pre-fermentation, the bacterial cells were retained by the pressure difference within the fermenter using the nested permeable filter structure inside the fermenter, and the clear enzymatic hydrolysate was collected and transferred to the main fermenter.

[0030] S32, Main fermentation section: Inoculate the clarified enzymatic hydrolysate with an inoculum size of 5 × 10⁻⁶. 7 CFU / mL of *Lactobacillus plantarum* and *Bifidobacterium breve* (inoculation ratio 2:1) were inoculated separately, and primary fermentation was carried out at 37°C. pH, lactic acid content, and viable cell count were monitored in real time. The fermentation was completed when the pH dropped to 4.2 and the total viable cell count reached 2.5 × 10⁻⁶. 9 Fermentation is considered complete when the CFU / mL and lactic acid content reach 5.2 g / L.

[0031] S4. Post-processing: The main fermentation broth was subjected to high-pressure microfluidic homogenization twice at 100 MPa, and the average particle size was found to be 450 nm. Subsequently, it was pasteurized at 85℃ for 15 minutes and then filled to obtain the finished product.

[0032] result: The resulting lily puree beverage was tested and found to have a residual sugar content of 0.3g / 100mL and a total viable bacteria count of 3.2×10⁻⁶. 9 The product has a CFU / mL concentration and a lactic acid content of 6.5 g / L. After standing at 25°C for 6 months, the product exhibits a uniform appearance with no layering or sedimentation.

[0033] in conclusion: This embodiment precisely controls carbon source release through three-stage gradient enzymatic hydrolysis, maximizes the synergistic function of microbial strains through segmented fermentation, and constructs a stable nanofiber network through high-pressure microfluidic homogenization, successfully preparing a sugar-free, high-live-bacteria, and highly stable lily pulp beverage, with all indicators exceeding the expected targets.

[0034] Example 2: This embodiment is intended to verify the feasibility of the technical solution of the present invention under the conditions of the lower limit of reducing sugar content and DE value boundary defined in the claims.

[0035] The steps are as follows: With all other preparation conditions the same as in Example 1, only the enzymatic hydrolysis endpoint control strategy in step S2 was adjusted. By reducing the amount of saccharifying enzyme added to 120 U / g and shortening the third-stage debranching hydrolysis time to 30 minutes, the reducing sugar content at the hydrolysis endpoint was controlled to be 4.5 g / 100 mL, at which point the DE value was measured to be 80.5%. The operating parameters for the remaining steps (S1, S3, S4) were completely consistent with those in Example 1.

[0036] result: The product, after testing, had a residual sugar content of 0.2 g / 100 mL and a total viable bacterial count of 2.8 × 10⁻⁶ after fermentation. 9 The product has a CFU / mL concentration and a lactic acid content of 5.8 g / L. After standing at 25°C for 6 months, no precipitation was observed, with only very slight optical wettability, indicating good overall stability.

[0037] in conclusion: The results show that even under the conditions that the reducing sugar content is at the lower limit of the claim range (4.5 g / 100 mL) and the DE value just reaches 80%, the technical solution of the present invention can still be successfully implemented, and the resulting product can still meet the technical requirements of sugar-free, high viable bacteria and stability, which proves the broad adaptability of the parameter range and the robustness of the solution.

[0038] Example 3: This embodiment is intended to verify the feasibility of the technical solution of the present invention under the upper limit of reducing sugar content and the boundary conditions of high-pressure homogenization as defined in the claims.

[0039] The steps are as follows: With all other preparation conditions the same as in Example 1, only the parameters of steps S2 and S4 were adjusted. In step S2, the amount of saccharifying enzyme added was increased to 180 U / g and the debranching hydrolysis time was extended to 50 minutes, controlling the reducing sugar content at the hydrolysis endpoint to 5.5 g / 100 mL and the DE value to 92%. In step S4, the high-pressure microfluidic homogenization pressure was adjusted to 120 MPa, and the number of homogenization cycles was still determined by particle size feedback control (resulting in 2 cycles). The operating parameters for the remaining steps were completely consistent with those in Example 1.

[0040] result: The product, after testing, had a residual sugar content of 0.4 g / 100 mL and a total viable bacterial count of 3.5 × 10⁻⁶ after fermentation. 9 The product has a CFU / mL concentration and a lactic acid content of 7.1 g / L. After standing at 25°C for 6 months, the product showed no stratification or precipitation, demonstrating excellent system stability.

[0041] in conclusion: The results show that the technical solution of the present invention remains effective even when the reducing sugar content is close to the upper limit (5.5 g / 100 mL) and a relatively high homogenization pressure (120 MPa). The higher initial sugar content provides a more sufficient carbon source for probiotics, resulting in a slight increase in the number of viable bacteria and lactic acid content. Furthermore, high-pressure homogenization further refines the fiber particle size, confirming that the parameters within the scope of the claims can achieve the beneficial effects of the present invention.

[0042] Example 4: This embodiment is intended to verify the boundary conditions of the pre-fermentation time and pH maintenance conditions defined in the claims.

[0043] The steps are as follows: With all other preparation conditions the same as in Example 1, only the parameters of step S31 were adjusted. The pre-fermentation time was shortened to 4 hours, and the pH was strictly controlled to be maintained at 5.0 (lower limit) during fermentation. The parameters of the main fermentation stage and other steps remained the same as in Example 1.

[0044] result: The product, after testing, had a residual sugar content of 0.35 g / 100 mL and a total viable bacterial count of 2.6 × 10⁻⁶ after fermentation. 9 The product has a CFU / mL concentration and a lactic acid content of 5.5 g / L. After standing at 25°C for 6 months, no obvious stratification was observed.

[0045] in conclusion: The results showed that even with the shortest pre-fermentation time of 4 hours and the pH maintained at the lower limit of 5.0, Bacillus amyloliquefaciens could still complete the basic substrate modification task, ensuring the smooth progress of the subsequent main fermentation, proving that the parameter range was feasible.

[0046] Example 5: This embodiment is intended to verify the boundary conditions of the main fermentation time and endpoint determination index as defined in the claims.

[0047] The steps are as follows: With all other preparation conditions the same as in Example 1, only the parameters of step S32 were adjusted. The main fermentation time was controlled at 8 hours (lower limit), and the pH was 4.5 with a total viable cell count of 1.0 × 10⁻⁶. 9 CFU / mL and lactic acid content of 4.0 g / L were used as the fermentation endpoint criteria. The remaining parameters were the same as in Example 1.

[0048] result: The obtained product, after testing, met all the above endpoint conditions. The residual sugar content of the finished product was 0.45 g / 100 mL, and the total viable bacteria count was 1.1 × 10⁻⁶. 9The product contains CFU / mL and has a lactic acid content of 4.1 g / L. After standing at 25°C for 6 months, a small amount of sediment appeared at the bottom, but it dispersed quickly after shaking, and the product was considered stable overall.

[0049] in conclusion: The results show that when the main fermentation time and endpoint indicators are at the lower limit of the scope defined in the claims, the product can still meet the basic requirements of sugar-free and live bacteria. Although the stability is slightly inferior to the preferred embodiment, it still falls within the scope of the technical effects protected by this invention, which confirms the effectiveness of the parameter range.

[0050] Example 6: This embodiment aims to verify the application effect of the adaptive determination method based on detection data analysis (claim 7) in the control of enzymatic hydrolysis endpoint.

[0051] The steps are as follows: In step S2, during the gradient enzymatic hydrolysis, an adaptive judgment system is activated. The sliding window length N=5, weighting factor β=0.85, and consecutive trigger count M=3 are set. The system collects the reducing sugar content RS_t and DE value D_t in real time, calculates the weighted moving average Z_t, and dynamically generates a threshold D_th based on the fluctuation of RS_t. When Z_t exceeds D_th three consecutive times and the RS_t readings are 5.1g / 100mL, 5.2g / 100mL, and 5.0g / 100mL respectively, the system automatically determines the end of the enzymatic hydrolysis and triggers the enzyme inactivation program. The remaining steps are the same as in Example 1.

[0052] result: The reducing sugar content of this batch of enzymatic hydrolysate was consistently controlled at 5.1 g / 100 mL, with a DE value of 89%. The residual sugar in the final product was 0.3 g / 100 mL, and the batch-to-batch coefficient of variation (CV) was less than 3%.

[0053] in conclusion: The results show that the adaptive judgment method can effectively smooth out detection noise, adapt to raw material fluctuations, accurately lock the enzymatic hydrolysis endpoint, and significantly improve the automation level of the production process and the consistency of product quality.

[0054] Example 7: This embodiment is intended to verify the application effect of the fermentation endpoint determination method based on multi-parameter fusion and time-series stabilization (claim 8).

[0055] The steps are as follows: In step S3, during the main fermentation process, a multi-parameter fusion judgment system is activated. Weighting coefficients w1=0.4, w2=0.4, w3=0.2 are set to construct a comprehensive fermentation index F_t. Asymmetric hysteresis intervals F_low=-0.1, F_high=0.2 are set. The system monitors pH, lactic acid content L_t, and the logarithmic number of viable cells C_t in real time. Fermentation is considered complete only when F_t is lower than F_low for three consecutive times. The remaining steps are the same as in Example 1.

[0056] result: During fermentation, there was a momentary drop in the pH probe reading due to bubbles from stirring, but F_t did not meet the condition of continuously being lower than F_low, and the system did not mistakenly terminate the process. Fermentation was ultimately terminated accurately when F_t remained consistently and stably below the threshold, and the final product specifications were comparable to those of Example 1.

[0057] in conclusion: The results show that this method effectively filters out sensor signal jitter, avoids misoperation caused by single-point data anomalies, and ensures accurate and reliable determination of the fermentation endpoint.

[0058] Example 8: This embodiment is intended to verify the application effect of the adaptive homogeneous control method based on particle size feedback (claim 9).

[0059] The steps are as follows: In step S4, during the high-pressure microfluidic homogenization process, the particle size feedback control system is activated. The target particle size is set to d_tar = 500 nm. After the first homogenization, d_1 is detected as 780 nm, and the PI controller predicts d_2_pred = 580 nm, so a second homogenization is performed. After the second homogenization, d_2 is detected as 540 nm, and d_3_pred is predicted as 480 nm. The system determines that a third homogenization is unnecessary and stops. The remaining steps are the same as in Example 1.

[0060] result: The final product has an average particle size of 540nm, meeting the requirement of ≤800nm. Compared with the traditional process of fixed three homogenizations, this embodiment saves 33% of homogenization energy consumption, and the product stability is not affected.

[0061] in conclusion: The results show that adaptive control based on particle size feedback can intelligently determine the number of homogenization cycles according to the actual state of the material, achieving energy saving and consumption reduction while ensuring product quality.

[0062] Example 9: This embodiment is intended to verify the effect of the built-in self-resonant enhanced cavitation structure nozzle (claim 10) on the homogenization effect.

[0063] The steps are as follows: In step S4, a homogenizing nozzle with a built-in Helmholtz resonant cavity and a spiral microstructure machined on its inner surface is used. Homogenization is performed at a pressure of 80 MPa, with other conditions the same as in Example 1.

[0064] result: Under 80 MPa pressure, only two homogenization cycles are required to achieve an average particle size of 490 nm, while a traditional straight-through nozzle requires three cycles to achieve a similar particle size under the same pressure. Furthermore, products made using this nozzle exhibit approximately 20% higher storage modulus compared to products made with traditional nozzles, and demonstrate better stability after 6 months of settling.

[0065] in conclusion: The results show that the specially structured nozzle significantly improves the fiber nanocompositing efficiency by enhancing the cavitation effect and strengthens the self-stabilizing network, verifying the unexpected technical effects brought about by this hardware improvement.

[0066] Example 10: This embodiment aims to compare the effects of different inoculation ratios of bacterial strains on the flavor and live bacteria composition of the product.

[0067] The steps are as follows: With all other preparation conditions the same as in Example 1, only the inoculation ratio of *Lactobacillus plantarum* to *Bifidobacterium breve* in step S32 was adjusted to 1:2. All other parameters remained unchanged.

[0068] result: The viable count ratio of *Lactobacillus plantarum* to *Bifidobacterium breve* in the obtained product was approximately 1:2.5. Sensory evaluation showed that at this ratio, the product had a slightly higher acetic acid content, a richer flavor profile, and a more balanced sweet and sour ratio. The residual sugar in the finished product was 0.3 g / 100 mL, and the total viable count was 3.0 × 10⁻⁶. 9 CFU / mL.

[0069] in conclusion: The results show that adjusting the strain ratio within the range of 1:5 to 5:1 as defined in the claims can effectively regulate the organic acid spectrum and flavor characteristics of the product, meet the taste needs of different consumer groups, and maintain the core probiotic function indicators.

[0070] Example 11: This embodiment is a performance and effect verification embodiment, which aims to comprehensively evaluate the significant progress of the technical solution of the present invention compared with the prior art by setting a comparative example.

[0071] Scale settings: Comparative Example 1 (Fermentation with Added Sugar): The steps of Example 1 were followed, but sucrose was added after enzymatic hydrolysis in S2 to make the total reducing sugar 12g / 100mL. The fine control of three-stage gradient enzymatic hydrolysis was not performed, and mixed culture fermentation was carried out directly (without a pre-fermentation stage). In S4, high-pressure homogenization was not performed, but 0.1% sodium carboxymethyl cellulose (CMC) was added as a stabilizer.

[0072] Comparative Example 2 (Single-step enzymatic hydrolysis and mixed fermentation): The steps of Example 1 were followed, but S2 only used a single-step saccharification and enzymatic hydrolysis (60℃, pH 4.5, 90 minutes) without distinguishing the three gradients; S3 did not perform segmented fermentation and directly inoculated the three strains at the same time for fermentation; S4 used 50MPa pressure to fix homogenize once.

[0073] Comparative Example 3 (missing key components): The steps of Example 1 were followed, but in S3, Lactobacillus plantarum and Bifidobacterium breve were not inoculated; only Bacillus amyloliquefaciens fermentation was performed.

[0074] Test results: The samples obtained in Examples 1-3 and Comparative Examples 1-3 were subjected to physicochemical, microbiological, and stability tests. The specific data are shown in Table 1.

[0075] Table 1 Performance test results of the examples and comparative examples Effect Analysis: As shown in Table 1, the products obtained in Examples 1-3 are significantly better than the comparative examples in terms of residual sugar content, total viable bacteria count, and stability.

[0076] First, regarding the sugar-free characteristic: Comparative Example 1, due to the addition of sucrose, had a residual sugar content as high as 8.5g / 100mL, which completely fails to meet the sugar-free standard; Comparative Example 2, due to insufficient single-step enzymatic hydrolysis, had a large amount of non-fermentable dextrin and some unmetabolized sugars remaining, with a residual sugar content of 1.2g / 100mL; while the embodiment of this invention, through three-stage gradient enzymatic hydrolysis, precisely controls the carbon source (4.0~6.0g / 100mL), so that the residual sugar is stably controlled below 0.5g / 100mL, truly achieving sugar-free fermentation.

[0077] Secondly, regarding viable cell count and flavor: Comparative Example 3, due to the absence of the main fermentation strain, had almost no viable cells and no lactic acid production; Comparative Example 2, due to competition among strains during simultaneous inoculation and low substrate conversion rate, had a viable cell count of only 5.0 × 10⁻⁶. 8 CFU / mL; however, the embodiments of this invention, through a segmented fermentation strategy and utilizing a pre-modified substrate of Bacillus amyloliquefaciens, significantly promoted the subsequent proliferation of probiotics, achieving a viable count as high as 10. 9It is at the CFU / mL level, rich in lactic acid, and has a pure flavor.

[0078] Finally, regarding stability: Although Comparative Example 1 added the chemical thickener CMC, it still showed severe stratification and precipitation after long-term standing, indicating that chemical additives have limited effects in complex fermentation systems; Comparative Example 2, due to the lack of effective homogenization, had a particle size as high as 1200nm, resulting in obvious stratification; while the embodiments of this invention, through high-pressure microfluidic homogenization, nano-sized dietary fiber (≤800nm) to construct a natural self-stabilizing network, achieving no stratification for 6 months without any chemical additives.

[0079] In this application embodiment, the test samples included the products prepared in each of Examples 1-10. Experimental results showed that the probiotic fermented sugar-free lily puree beverage prepared according to this invention exhibited good effects in regulating intestinal peristalsis and restoring flora balance in animal constipation models and intestinal flora imbalance models. Furthermore, because it contains no exogenous sugars or chemical additives, long-term consumption does not impose a metabolic burden. Therefore, this product can be used to prepare food, health products, or pharmaceutical preparations for the prevention and / or treatment of functional constipation, intestinal flora imbalance, and related metabolic syndromes. This application conclusion is based on the high viable bacteria count (≥1×10⁻⁶) confirmed in the aforementioned performance and effect examples. 9 The data (CFU / mL), abundant lactic acid content, and excellent stability were reasonably derived and did not introduce any unproven new uses.

Claims

1. A method for preparing a probiotic-fermented sugar-free lily puree beverage, characterized in that, Includes the following steps: S1. Raw material processing: The lily bulb raw materials are washed, sliced, and treated for color protection. Then, 1 to 3 times the weight of purified water is added and pulped to obtain lily pulp. S2. Gradient enzymatic hydrolysis: The lily slurry is subjected to a three-stage gradient enzymatic hydrolysis to convert the starch in it into fermentable sugars. The hydrolysis endpoint is achieved when the reducing sugar content is 4.0~6.0g / 100mL. S3. Segmented Fermentation: The lily enzymatic hydrolysate obtained after enzymatic hydrolysis is cooled to 35-40℃, and the pH is adjusted to 6.0-7.

0. The segmented fermentation includes: S31, Pre-fermentation section: Bacillus amyloliquefaciens is inoculated into the lily enzymatic hydrolysate, and fermentation is carried out at 35~40℃; S32. Main fermentation stage: After the pre-fermentation is completed, a compound bacterial agent containing Lactobacillus plantarum and Bifidobacterium breve is inoculated into the system, and the main fermentation is carried out at 35~40℃ until the pH of the fermentation broth drops to 3.8~4.

5. S4. Post-processing: After the main fermentation is completed, the fermentation liquid is subjected to high-pressure micro-jet homogenization at a pressure of 60~150MPa, and then sterilized to obtain the probiotic fermented sugar-free lily puree beverage.

2. The preparation method according to claim 1, characterized in that, The three-stage gradient enzymatic hydrolysis includes: First stage of enzymatic hydrolysis: Add α-amylase and carry out enzymatic hydrolysis at 60~75℃; The second stage of enzymatic hydrolysis: add saccharifying enzyme and carry out enzymatic hydrolysis at 55~65℃ and pH 4.0~5.5; The third stage of enzymatic hydrolysis: add debranching enzyme and carry out enzymatic hydrolysis at 55~65℃.

3. The preparation method according to claim 2, characterized in that, The α-amylase is selected from at least one of mesophilic α-amylase, thermostable α-amylase, and bacterial α-amylase; the saccharifying enzyme is selected from at least one of glucoamylase, β-amylase, and fungal saccharifying enzyme; and the debranching enzyme is selected from at least one of pullulanase and isoamylase.

4. The preparation method according to claim 1, characterized in that, In the gradient enzymatic hydrolysis, the reducing sugar content at the end of the hydrolysis is 4.5~5.5g / 100mL, and the DE value of the lily hydrolysate is ≥80%.

5. The preparation method according to claim 1, characterized in that, In the segmented fermentation, the fermentation time of the pre-fermentation stage is 4 to 10 hours, and the pH is maintained at ≥5.0 during the fermentation process; the fermentation time of the main fermentation stage is 8 to 30 hours.

6. The preparation method according to claim 5, characterized in that, The main fermentation stage ends when all three of the following conditions are met: the pH of the fermentation broth is 4.0-4.5, and the total viable count is ≥1×10⁻⁶. 9 CFU / mL, lactic acid content ≥4.0g / L.

7. The preparation method according to claim 1, characterized in that, The gradient enzymatic hydrolysis in step S2 further includes an adaptive determination of the hydrolysis endpoint based on detection data analysis. The determination method includes: real-time monitoring of the reducing sugar content RS_t and DE value D_t during the hydrolysis process; constructing a discrete sliding window of length N and calculating the weighted moving average Z_t of D_t within the window; calculating the standard deviation of RS_t within the sliding window as a fluctuation index σ, and dynamically generating an adaptive DE value determination threshold D_th according to D_th=D_ref-k*σ (where D_ref is a preset DE target value and k is a preset correction coefficient); when Z_t exceeds D_th for M consecutive times and RS_t is within the range of 4.5~5.5g / 100mL, the hydrolysis endpoint is determined.

8. The preparation method according to claim 1, characterized in that, The main fermentation in step S3 also includes determining the fermentation endpoint based on multi-parameter fusion and time-series debouncing. The determination method includes: real-time monitoring of the pH value, lactic acid content L_t, and logarithmic number of viable bacteria C_t of the fermentation broth during the main fermentation process; constructing a comprehensive fermentation index F_t=w1A+w2B+w3*C, where A is a pH-related term, B is a lactic acid-related term, C is a viable bacteria-related term, and w1, w2, and w3 are weighting coefficients whose sum is 1; setting an asymmetric hysteresis interval [F_low, F_high], where fermentation is determined to end when F_t is continuously lower than F_low multiple times, and continuous fermentation is determined when F_t is continuously higher than F_high multiple times.

9. The preparation method according to claim 1, characterized in that, The high-pressure microjets homogenization in step S4 further includes adaptive control of the number of homogenization cycles based on particle size feedback. The control method includes: after each homogenization process, detecting the average particle size d_n of the fermentation broth; using the target particle size d_tar as a set value, calculating the current particle size deviation e_n = d_n - d_tar, and accumulating the deviation integral S_n; based on the discrete proportional-integral control law, predicting the particle size d_{n+1}pred after the next homogenization, and deciding whether to continue the next homogenization cycle based on the relationship between d_{n+1}pred and d_tar.

10. The preparation method according to claim 1, characterized in that, The high-pressure micro-jet homogenization in step S4 uses a homogenizing nozzle with a built-in self-resonant enhanced cavitation structure. The nozzle's outlet front end is provided with a Helmholtz resonant cavity, and the inner surface of the resonant cavity is processed with microstructures to guide the fluid to generate swirling flow and excite the cavitation effect.

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