Preparation method of grapefruit juice based on full-fruit debittering microencapsulation

Abstract

The present application relates to the technical field of food processing, and in particular to a method for preparing grapefruit juice based on whole fruit debittering microencapsulation. The method separates the grapefruit peel and pulp, respectively, and then makes pulp; the peel pulp is fermented by Aspergillus niger and Lactobacillus plantarum to remove bitterness; the debittered peel pulp is subjected to enzymatic modification by complex enzyme preparation and ultrasonic treatment; the pulp is clarified by pectinase enzymolysis; the grapefruit peel essential oil is prepared into gradient microencapsulated essential oil microcapsules by primary embedding with β-cyclodextrin and secondary embedding with sodium alginate-calcium chloride; finally, the modified peel pulp, clarified pulp juice and gradient microencapsulated essential oil microcapsules are mixed and adjusted, and then homogenized, degassed and sterilized to obtain the finished product. The present application realizes the high-value utilization of whole grapefruit, and through the synergistic effect of fermentation debittering, enzymatic modification and gradient microencapsulation, the prepared grapefruit juice has very low bitterness, smooth taste, persistent aroma and high dietary fiber content, and has good application prospect.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, specifically to a method for preparing grapefruit juice based on whole-fruit debittering microencapsulation. Background Technology

[0002] The pomelo processing industry has long faced a dilemma: on the one hand, pomelo peel accounts for 40%–50% of the fruit's total weight and is rich in beneficial components such as essential oils, dietary fiber, and pectin, whose nutritional and economic value has long been recognized in the field; on the other hand, existing pomelo juice preparation technologies generally employ the method of peeling and extracting the pulp, using only the pulp for juicing, while the peel is mostly treated as waste. Although existing technologies have attempted to mix the peel with the juice after pulping, or to ferment the peel with lactic acid bacteria to remove bitterness and then produce a separate peel pulp product, these solutions either simply add the peel directly after pulping, resulting in a product that is "slightly sour and bitter" and has an unpleasant taste, or they process the peel and pulp separately and then simply mix them, resulting in insufficient integration of the peel pulp and juice and poor product uniformity. The reason for this is that the bitter substances in pomelo peel are much higher than in the pulp, the coarse fiber tissue is dense, and the essential oil components can produce a pungent taste if not properly processed. The combination of these three factors makes it difficult to achieve a taste level acceptable to consumers when utilizing the whole fruit. Existing methods such as resin adsorption and β-cyclodextrin encapsulation only achieve a removal rate of about 47% for limonene. How to fully utilize the beneficial components of essential oils and dietary fiber in the peel while systematically addressing the bitterness, rough texture, and aroma imbalance caused by the peel, so that whole-fruit grapefruit juice truly possesses commercially viable taste and flavor quality, has become a critical technological bottleneck that urgently needs to be overcome in this field. Summary of the Invention

[0003] The technical problem to be solved by this invention is: how to fully utilize the beneficial components of essential oils and dietary fiber in the peel while systematically solving the problems of bitterness, rough texture and aroma incompatibility caused by the peel, so that whole grapefruit juice can truly have commercially viable taste and flavor quality.

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows: A method for preparing grapefruit juice based on whole-fruit debittering microencapsulation includes the following steps: Step 1: Pre-treatment of whole pomelo: Separate the pomelo peel from the pulp. Collect the oil cell layer and white peel layer of the peel separately. Remove the seeds from the pulp. Extract pomelo peel essential oil from the oil cell layer peel. Combine the extracted oil cell layer residue with the white peel layer peel, cut into pieces, add water and blend into a pulp to obtain a coarse peel pulp. Add water to the pulp and blend into a pulp to obtain a coarse pulp pulp. Step 2: Debittering by microbial directional fermentation of fruit peel: Mix Aspergillus niger and Lactobacillus plantarum at a live cell ratio of 1:1 to 3:1 to prepare a compound fermentation inoculum. Add the compound fermentation inoculum to the crude fruit peel pulp obtained in Step 1. The inoculation amount is 2% to 6% of the mass of the crude fruit peel pulp. Perform anaerobic-aerobic two-stage fermentation at 28 to 32°C. After fermentation, sterilize and cool to obtain debittered fruit peel pulp. Step 3: Enzymatic hydrolysis and synergistic modification of fruit peel dietary fiber: A compound enzyme preparation, consisting of cellulase, pectinase and xylanase, is added to the debittered fruit peel pulp obtained in Step 2. The enzymatic hydrolysis reaction is carried out at 45-55℃ and pH 4.5-5.5 for 60-180 minutes. Ultrasonic treatment is used during the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, the enzyme is inactivated and cooled to obtain the modified fruit peel pulp. Step 4: Enzymatic hydrolysis and clarification of fruit pulp: Add pectinase to the crude fruit pulp obtained in Step 1, and hydrolyze for 30 to 90 minutes at 45-50℃ and pH 3.5-4.5. After inactivating the enzyme, centrifuge and collect the supernatant to obtain clarified fruit pulp juice. Step 5: Gradient microencapsulation of grapefruit peel essential oil: The grapefruit peel essential oil obtained in Step 1 is subjected to primary microencapsulation and secondary microencapsulation in sequence. The primary microencapsulation uses β-cyclodextrin as the wall material for encapsulation, and the secondary microencapsulation uses sodium alginate-calcium chloride system for solidification and encapsulation, to obtain gradient microencapsulated essential oil microcapsules. Step 6: Whole-fruit grapefruit juice preparation and microencapsulation integration: Mix the modified peel pulp obtained in Step 3 and the clarified pulp juice obtained in Step 4, add the gradient microencapsulated essential oil microcapsules obtained in Step 5, homogenize, deaerate, sterilize, and fill to obtain the finished grapefruit juice based on whole-fruit debittering microencapsulation.

[0005] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, in step 1, the peel is cut into pieces and water is added at a material-to-liquid ratio of 1:2 to 1:5, and then pulped at 40 to 50°C; the pulp is pulped at a material-to-liquid ratio of 1:1 to 1:3.

[0006] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the total viable bacteria concentration of the compound fermentation inoculum in step 2 is 10. 8 ~10 9 CFU / mL; the two-stage fermentation consists of anaerobic fermentation for 12-24 hours followed by aerobic fermentation with sterile air for 6-12 hours, for a total fermentation time of 18-36 hours.

[0007] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, in step 2, 0.5% to 2.0% of the mass of the crude peel pulp is added to the crude peel pulp, wherein the carbon source is one or more of glucose, sucrose, or fructooligosaccharides; and the fermentation pH is 5.0 to 5.5.

[0008] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the enzyme activity ratio of cellulase, pectinase and xylanase in the compound enzyme preparation in step 3 is 2:1:1, and the total amount of compound enzyme preparation added is 0.1% to 0.5% of the mass of the debittered fruit peel pulp.

[0009] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the ultrasonic treatment in step 3 has a power of 200-400W and is performed intermittently, with an interval of 2-5 minutes between ultrasonic treatments of 5-10 minutes.

[0010] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the specific method of primary microencapsulation in step 5 is as follows: β-cyclodextrin is dissolved in water at 40-50°C, grapefruit peel essential oil is added under stirring conditions, the mass ratio of essential oil to β-cyclodextrin is 1:8 to 1:15, and the mixture is stirred and encapsulated at 50-60°C for 1-2 hours. After cooling, standing, filtering, washing, and drying, primary essential oil microcapsules are obtained.

[0011] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the specific method of secondary microencapsulation in step 5 is as follows: the primary essential oil microcapsules are dispersed in a sodium alginate solution with a mass fraction of 1% to 3%, and then dropped into a calcium chloride solution with a mass fraction of 2% to 5% through a sharp-pore coagulation bath method to solidify for 30 to 60 minutes. After filtration, washing, and drying, gradient microencapsulated essential oil microcapsules are obtained.

[0012] Furthermore, in the above-mentioned method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, the mixing mass ratio of the modified peel pulp to the clarified pulp juice in step 6 is 1:3 to 2:3; the amount of gradient microencapsulated essential oil microcapsules added is 0.5% to 2.0% of the mass of the whole-fruit grapefruit juice base material; the homogenization pressure is 20 to 25 MPa, and the homogenization is performed 1 to 2 times.

[0013] The beneficial effects of this invention are as follows: By constructing a three-in-one whole-fruit utilization technology system of "microbial directional fermentation for debittering—enzymatic hydrolysis synergistic modification—gradient microencapsulation," this invention breaks the long-standing technical mindset of "removing the peel and extracting the pulp" in the field, and realizes the high-value integration of beneficial components such as essential oils and dietary fiber in grapefruit peel into juice products. It is well known in the art that the content of bitter substances such as naringin in grapefruit peel is much higher than in the pulp, and the coarse fiber tissue is dense. Direct utilization inevitably leads to an unacceptable bitter taste and roughness, which is precisely the technical bottleneck restricting whole-fruit processing. This invention does not simply mechanically superimpose fermentation debittering, enzymatic hydrolysis modification, and microencapsulation. Instead, based on a profound understanding of the intrinsic connections between each step, it designs a specific time sequence of fermentation followed by enzymatic hydrolysis. First, through a two-stage anaerobic-aerobic fermentation using a compound microbial strain, the metabolic synergy of Aspergillus niger and Lactobacillus plantarum solves the technical problem of complete debittering by a single method. The enzymatic hydrolysis modification on this basis cleverly utilizes the initial softening effect of fermentation on the fruit peel tissue, enabling the compound enzyme preparation to more efficiently convert insoluble dietary fiber into soluble dietary fiber. Furthermore, the trace bitter substances remaining in the system after fermentation are further transformed during enzymatic hydrolysis. The two produce a synergistic effect of "debittering first to promote enzymatic hydrolysis, and enzymatic hydrolysis assisting in debittering". Meanwhile, this invention performs gradient microencapsulation of grapefruit peel essential oil and adds it during the blending stage, avoiding the inhibitory effect of terpenoid compounds in the essential oil on microbial fermentation and enzymatic activity. This not only preserves the nutritional value of the whole grapefruit but also achieves a refreshing and smooth drinking experience, fundamentally solving the common industry problem of the difficulty in achieving both high-value utilization of grapefruit peel and excellent taste. Detailed Implementation

[0014] To explain in detail the technical content, objectives, and effects of the present invention, the following description is provided in conjunction with the embodiments.

[0015] The Aspergillus niger used in this invention ( Aspergillus niger This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), and belongs to the existing strains, with the accession number CGMCC No. 3.3928. It is available to the public from this collection center. This strain can secrete naringinase, which has the activity of hydrolyzing naringin.

[0016] The Lactobacillus plantarum used in this invention ( Lactobacillus plantarum This strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), and belongs to the existing strains, with the accession number CGMCC No. 1.557. It is available to the public from this collection center. During its metabolism, this strain can degrade limonene-like bitter substances and produce organic acids and flavor compounds, which helps to improve the flavor of fruit peels.

[0017] Both of the above-mentioned strains are commonly used microbial strains in the food processing field and can be activated, propagated, and preserved using conventional methods.

[0018] Example 1 A method for preparing grapefruit juice based on whole-fruit debittering microencapsulation includes the following steps: Step 1: Pre-treatment of whole grapefruit; Take 100kg of fresh, ripe pomelos, remove any rotten or spoiled fruit, and wash the surface with running water to remove dirt. After washing, separate the peel from the pulp and collect them separately. This batch of raw materials yielded approximately 42kg of peel and approximately 52kg of pulp after removing the seeds.

[0019] The pericarp was further separated into the oil cell layer and the albedo layer. The oil cell layer pericarp was cut into small cubes of approximately 1 cm and the essential oil was extracted using a cold pressing method. The specific procedure was as follows: the small cubes of oil cell layer pericarp were fed into a screw press and pressed at 10–15 MPa at room temperature. The press liquid was collected and centrifuged at 3000 rpm for 10 minutes. The upper oil phase was taken as grapefruit peel essential oil. This batch yielded approximately 85 mL (approximately 77 g) of essential oil. The residue from the oil cell layer after pressing was combined with the albedo layer pericarp.

[0020] After removing the seeds, add water to the pulp at a ratio of 2L of purified water per 1kg of pulp, and then grind it using a colloid mill with the gap adjusted to 0.8mm and the speed at 3000rpm. Circulate the pulping process twice to obtain approximately 156kg of pulp pulp.

[0021] Step 2: Targeted microbial fermentation of fruit peels to remove bitterness; First, prepare the compound fermentation inoculum. Then, add *Aspergillus niger* (…). Aspergillus niger CGMCC No. 3.3928 was inoculated onto PDA slant medium (formulation: potato 200 g / L, glucose 20 g / L, agar 15 g / L, pH natural) and activated by incubation at 30°C for 72 hours. The spores on the slant were eluted with sterile physiological saline containing 0.1% (v / v) Tween-80 to prepare a spore suspension, which was then adjusted to a concentration of 10. 7 Spores / mL. Take the spore suspension and transfer it to PDA liquid medium (without agar) at an inoculation rate of 5% (volume fraction). Incubate at 30°C and 150 rpm for 48 hours until the logarithmic growth phase.

[0022] Lactobacillus plantarum ( Lactobacillus plantarumCGMCC No. 1.557 was inoculated into MRS liquid medium (formulation: peptone 10 g / L, beef extract 10 g / L, yeast extract 5 g / L, glucose 20 g / L, Tween-80 1 mL / L, diammonium citrate 2 g / L, sodium acetate 5 g / L, magnesium sulfate 0.58 g / L, manganese sulfate 0.25 g / L, dipotassium hydrogen phosphate 2 g / L, pH 6.2–6.4), and activated by static incubation at 37°C for 24 hours, followed by two consecutive subcultures. The activated bacterial culture was then inoculated at 2% (v / v) into fresh MRS medium and incubated at 37°C for 18 hours until the logarithmic growth phase.

[0023] Culture media of both bacteria were collected separately, and the bacterial pellets were collected by centrifugation at 3000 rpm for 10 minutes each time. The supernatant was discarded. The pellets were washed twice with sterile physiological saline (each time, an equal volume of sterile physiological saline was added, the pellets were resuspended by pipetting, and centrifuged again at 3000 rpm for 10 minutes). Finally, the pellets were resuspended in sterile physiological saline to the desired volume. After counting with a hemocytometer, Aspergillus niger and Lactobacillus plantarum viable cells were mixed at a ratio of 2:1 and diluted with sterile physiological saline to a total viable cell concentration of 5 × 10⁻⁶. 8 The CFU / mL concentration yields the composite fermentation culture. All the above operations were performed under aseptic conditions. This concentration is around 10. 8 ~10 9 Within the CFU / mL range, it falls into the middle value of that range.

[0024] Add 5.04 kg of the above-mentioned compound fermentation inoculum to 168 kg of the crude pulp of pericarp obtained in step 1, with the inoculum amount equivalent to 3% of the mass of the crude pulp. Simultaneously add 1.68 kg of anhydrous glucose, equivalent to 1.0% of the mass of the crude pulp. Glucose was chosen as the carbon source in this embodiment because it can be directly and rapidly utilized by *Aspergillus niger* and *Lactobacillus plantarum*, which is beneficial for the rapid proliferation of the inoculum and the accumulation of metabolites in the early stages of fermentation. Adjust the pH to 5.2 using a 1 mol / L citric acid solution.

[0025] Fermentation was carried out in a 5L mechanically stirred fermenter with a liquid volume of 3L (this example is a small-scale test; in actual production, it can be scaled up to an industrial fermenter). The fermentation temperature was controlled at 30℃, and the stirring speed was 100rpm. Fermentation employed a two-stage model: first, anaerobic fermentation was carried out at 30℃ and 100rpm for 18 hours without air introduction; then, sterile air was introduced at a flow rate of 1.0 vvm (i.e., the ratio of air volume introduced per minute to fermentation liquid volume was 1.0), and aerobic fermentation was carried out at 30℃ and 150rpm for 8 hours, for a total fermentation time of 26 hours. The two-stage fermentation strategy was designed based on the following: the anaerobic stage is conducive to the proliferation of *Lactobacillus plantarum* and the degradation of limonene-like substances, while the aerobic stage is conducive to the metabolic activity of *Aspergillus niger* and the secretion of naringinase; the sequential arrangement of the two stages maximizes the metabolic activity of both strains.

[0026] During fermentation, samples were taken every 6 hours to measure the pH change and naringin content of the fermentation broth. The fermentation endpoint was determined by the rate of change in naringin content: fermentation was terminated when the rate of change in naringin content measured in two consecutive samples (6 hours apart) was less than 5%. A debittering rate of naringin of 90% or higher could be used as a supplementary reference. After fermentation, the fermenter was heated to 90°C and pasteurized for 15 minutes, then cooled to room temperature to obtain approximately 162 kg of debittered fruit peel pulp.

[0027] Step 3: Enzymatic hydrolysis and synergistic modification of dietary fiber in fruit peel; Prepare the compound enzyme preparation. Take cellulase (10000 U / g), pectinase (50000 U / g), and xylanase (20000 U / g) and mix them evenly at an enzyme activity ratio of 2:1:1. Dissolve the compound enzyme preparation in a small amount of pH 5.0 phosphate buffer (0.05 mol / L, preparation method: weigh 6.80 g of potassium dihydrogen phosphate, dissolve it in pure water and make up to 1000 mL, then adjust the pH to 5.0 with 1 mol / L NaOH) to prepare a 10% (w / w) enzyme solution for later use.

[0028] Take 162 kg of the debittered fruit peel pulp obtained in step 2 and place it in an enzymatic hydrolysis reactor equipped with a stirrer, a heating and temperature control device, and an ultrasonic generator. Add the above enzyme solution so that the final amount of the compound enzyme preparation added reaches 0.2% of the mass of the debittered fruit peel pulp, that is, 0.324 kg of the actual compound enzyme preparation added. Adjust the pH to 5.0 with 1 mol / L citric acid or 1 mol / L NaOH.

[0029] The enzymatic hydrolysis conditions were controlled as follows: temperature 50℃, pH 5.0, stirring speed 200 rpm, and reaction time 120 minutes. An ultrasonic generator was activated simultaneously with the enzymatic hydrolysis reaction, using a frequency of 40 kHz and a power of 300 W. An intermittent treatment method was adopted, i.e., an 8-minute ultrasonic treatment followed by a 3-minute interval, repeated until the enzymatic hydrolysis was completed. The purpose of ultrasonic treatment was to disrupt the cell wall structure of the pericarp through cavitation, increasing the contact area between the enzyme and the substrate, thereby improving the enzymatic hydrolysis efficiency.

[0030] After the enzymatic hydrolysis reaction was completed, the reactor was quickly heated to 95°C and kept at that temperature for 8 minutes to inactivate the enzyme. Then it was cooled to room temperature to obtain approximately 158 kg of modified fruit peel pulp.

[0031] Step 4: Clarify the pulp using enzymatic hydrolysis; Take 156 kg of the crude pulp obtained in step 1, place it in an enzymatic hydrolysis tank, and add 0.078 kg of pectinase (enzyme activity 50000 U / g). The amount of pectinase used is equivalent to 0.05% of the mass of the crude pulp. Adjust the pH to 4.0 with 1 mol / L citric acid.

[0032] The enzymatic hydrolysis conditions were: temperature 48℃, pH 4.0, stirring speed 150 rpm, and reaction time 60 minutes.

[0033] After enzymatic hydrolysis, the hydrolysis tank was heated to 88℃ and held at this temperature for 8 minutes to inactivate the enzymes. After cooling to room temperature, it was centrifuged at 4000 rpm for 15 minutes, and the supernatant was collected, which is the clarified pulp juice. This batch yielded approximately 118 kg. The purpose of separately enzymatically hydrolyzing and clarifying the pulp is to avoid interference from pectin and other components in the pulp with the peel modification process. If the pulp and peel are mixed and processed together, the soluble sugars in the pulp will be preferentially utilized by the fermentation bacteria, affecting the fermentation and debittering efficiency of the peel.

[0034] Step 5: Gradient microencapsulation of grapefruit peel essential oil; The first step was the primary encapsulation of β-cyclodextrin. Approximately 0.62 kg of β-cyclodextrin (to achieve a β-cyclodextrin to oil mass ratio of approximately 1:8, specifically 0.616 kg) was dissolved in approximately 12 kg of purified water at 45°C (to achieve a β-cyclodextrin mass fraction of approximately 5%), and stirred until completely dissolved. Under stirring at 500 rpm, 85 mL (approximately 77 g) of the grapefruit peel essential oil obtained in step 1 was slowly injected into the β-cyclodextrin solution at a rate of 0.5 mL / min using a micro-injection pump. The slow injection was to ensure sufficient dispersion and encapsulation of the essential oil, avoiding uneven encapsulation due to excessively high local concentrations. After injection, the mixture was incubated in a 55°C water bath at 300 rpm for 1.5 hours with constant temperature stirring. After encapsulation, the mixture was allowed to cool naturally to room temperature and then placed in a 4°C refrigerator overnight (approximately 12 hours) to promote microcapsule release. After standing, the mixture was filtered (using Whatman No.1 filter paper). The filter cake was washed three times with a small amount of purified water and dried in an oven at 40°C until constant weight was achieved, yielding approximately 0.56 kg of first-grade essential oil microcapsules.

[0035] Next is the sodium alginate-calcium chloride secondary encapsulation. 0.15 kg of sodium alginate was dissolved in approximately 15 L of purified water to achieve a sodium alginate mass fraction of 1%. The solution was stirred at room temperature until completely dissolved (approximately 2 hours), and then allowed to stand to remove bubbles. 0.56 kg of the aforementioned primary essential oil microcapsules were dispersed in the sodium alginate solution and sheared at 8000 rpm for 5 minutes using a high-speed shear emulsifier to ensure uniform dispersion of the microcapsules.

[0036] Two-stage encapsulation was performed using a sharp-orifice-coagulation bath method. Using a syringe needle with an inner diameter of 0.8 mm, the above mixture was added dropwise at a rate of 2 mL / min to a 3% (w / w) calcium chloride solution. The volume of the calcium chloride solution was three times the volume of the mixture (approximately 47 L). The needle was held approximately 10 cm above the liquid surface, and the mixture was stirred slowly at 100 rpm during the addition. After the addition was complete, the mixture was allowed to solidify at room temperature for 45 minutes. The microcapsules were collected by filtration, washed three times with purified water, and dried in a 40°C oven to constant weight, yielding approximately 0.65 kg of gradient microencapsulated essential oil microcapsules.

[0037] The principle of gradient microencapsulation design lies in the following: primary encapsulation with β-cyclodextrin utilizes its molecular cavity structure to encapsulate essential oil molecules, achieving initial fixation and protection of the essential oil; secondary encapsulation with sodium alginate and calcium chloride further enhances the mechanical strength and sustained-release properties of the microcapsules through the external gel layer formed by calcium ion cross-linking. This dual-layer microencapsulation structure enables the essential oil to exhibit a two-stage release pattern—first sustained release, then sustained release—during juice storage and consumption.

[0038] Step 6: Whole-fruit grapefruit juice preparation and microencapsulation integration; Take 50 kg of the modified peel pulp obtained in step 3 and 150 kg of the clarified pulp juice obtained in step 4, with a mass ratio of 1:3, within the range of 1:3 to 2:3. Add both to a mixing tank and stir at 200 rpm for 15 minutes to mix them evenly, to obtain 200 kg of whole grapefruit juice base.

[0039] Add 2.0 kg of the gradient microencapsulated essential oil microcapsules obtained in step 5 to the above base material. The amount of microcapsules added is equivalent to 1.0% of the mass of the base material. Continue stirring at 150 rpm for 10 minutes to make the microcapsules evenly dispersed.

[0040] The prepared liquid mixture was fed into a high-pressure homogenizer and homogenized twice at a pressure of 22 MPa. The homogenized liquid mixture was then fed into a vacuum degassing tank and degassed for 15 minutes at a vacuum of 0.06–0.08 MPa and a temperature of 40°C.

[0041] The degassed liquid was filled into glass bottles (250 mL per bottle), sealed, and pasteurized in a 92°C water bath for 20 minutes. After pasteurization, it was cooled in stages: first in a cold water bath to 40°C, and then at room temperature to obtain the finished grapefruit juice product based on whole-fruit debittering microencapsulation. This batch of finished product weighed approximately 195 kg.

[0042] Comparative Example 1 The scheme of Example 1 differs in that the peel is not used. All grapefruit peels are discarded, and only the pulp (52kg) after removing the seeds is taken and mixed with water at a material-to-liquid ratio of 1:2 (to obtain approximately 156kg of crude pulp). The peel separation, essential oil extraction, and peel pulping in step 1 are not performed, the peel fermentation and debittering in step 2 are not performed, the peel enzymatic modification in step 3 is not performed, and the essential oil microencapsulation in step 5 is not performed (there is no source of essential oil). In step 6, only the clarified pulp juice obtained in step 4 is used directly for blending (without adding modified peel pulp and microencapsulated essential oil). The remaining operations (homogenization, degassing, sterilization, etc.) are exactly the same as in Example 1.

[0043] Comparative Example 2 The scheme in Example 1 differs in that the compound fermentation inoculum in step 2 is replaced with a single Aspergillus niger (CGMCC No. 3.3928) inoculum, without the addition of Lactobacillus plantarum. The preparation method of the single Aspergillus niger inoculum is exactly the same as the Aspergillus niger culture method in Example 1, and the total viable cell concentration is also adjusted to 5 × 10⁻⁶. 8 The inoculation rate was CFU / mL, and the inoculation amount remained at 3% (5.04 kg) of the peel pulp mass. The remaining steps (enzymatic modification, pulp enzymatic hydrolysis clarification, gradient microencapsulation, formulation, etc.) were exactly the same as in Example 1.

[0044] Comparative Example 3 The scheme in Example 1 differs in that step 3, which involves enzymatic hydrolysis and synergistic modification of the fruit peel dietary fiber, is omitted. The debittered fruit peel pulp, after fermentation and sterilization, is not subjected to enzymatic hydrolysis or ultrasonic treatment, and no compound enzyme preparation is added; it is directly used for subsequent formulation in step 6. The remaining steps (pretreatment, fermentation, pulp enzymatic hydrolysis and clarification, gradient microencapsulation, formulation, etc.) are exactly the same as in Example 1.

[0045] Comparative Example 4 The scheme of Example 1 differs in that step 5, the gradient microencapsulation of grapefruit peel essential oil, is omitted. The grapefruit peel essential oil extracted in step 1 is not subjected to primary β-cyclodextrin encapsulation and secondary sodium alginate-calcium chloride encapsulation. Instead, in step 6, during preparation, 85 mL (approximately 77 g) of grapefruit peel essential oil is directly added to the whole-fruit grapefruit juice base. The addition amount is 0.037% (77 g / 210 kg × 100%) of the base mass based on the essential oil weight. After stirring evenly, subsequent operations such as homogenization, degassing, and sterilization are performed. All other steps are exactly the same as in Example 1.

[0046] Comparative Example 5 The scheme in Example 1 differs in that the order of steps 2 (directed microbial fermentation for debittering of fruit peel) and 3 (enzymatic hydrolysis and synergistic modification of dietary fiber in fruit peel) is reversed. Specifically, the crude fruit peel pulp is first subjected to enzymatic hydrolysis and synergistic modification (0.324 kg of compound enzyme preparation is added, treated at 50°C, pH 5.0, with ultrasound assistance for 120 minutes, followed by enzyme inactivation at 95°C for 8 minutes), and then cooled before undergoing microbial fermentation for debittering (5.04 kg of compound fermentation inoculum is added, fermented at 30°C in two stages for a total of 26 hours, followed by pasteurization at 90°C for 15 minutes). Subsequent steps (enzymatic hydrolysis and clarification of fruit pulp, gradient microencapsulation, formulation, etc.) are exactly the same as in Example 1.

[0047] Analytical and testing methods: I. Determination of naringin content; 1. Instruments and Reagents: The high-performance liquid chromatograph (HPLC) is equipped with a UV detector (or diode array detector). Specific models are not limited; any chromatograph with equivalent detection performance can be used. The chromatographic column is a C18 column packed with octadecylsilane-bonded silica gel, with dimensions of 250 mm × 4.6 mm and a particle size of 5 μm. The mobile phase consists of chromatographically pure acetonitrile and analytically pure acetic acid; water is ultrapure water. Naringin standards are purchased from the China National Institutes for Food and Drug Control, with a purity of not less than 98%. The batch number is not limited; any calibrated and qualified standards can be used. Methanol used for sample pretreatment is chromatographically pure, and ultrapure water is used for extraction.

[0048] 2. Preparation of Standard Stock Solution: Accurately weigh 10.0 mg of naringin standard and place it in a 10 mL volumetric flask. Add methanol to dissolve and dilute to the mark. Stopper the flask and shake thoroughly to obtain a standard stock solution with a concentration of 1.0 mg / mL. Transfer the stock solution to a stoppered glass bottle and store at 4°C protected from light. The shelf life is one month.

[0049] 3. Accurately measure an appropriate amount of the standard stock solution and dilute it stepwise with methanol. Specifically, accurately measure 1.0 mL of the standard stock solution into a 10 mL volumetric flask, add methanol to the mark, and mix well to obtain an intermediate solution with a concentration of 100 μg / mL. Then, accurately measure appropriate amounts of the intermediate solution to prepare a series of standard working solutions with concentrations of 5, 10, 25, 50, 100, and 200 μg / mL. A calibrated pipette or pipette must be used for each concentration point preparation to ensure precision.

[0050] 4. Establishment of Standard Curve: Inject 10 μL of each of the above series of standard working solutions sequentially into the high-performance liquid chromatograph (HPLC), and record the peak area corresponding to each concentration. Inject twice at each concentration point and take the average peak area. Perform linear regression analysis with the standard working solution concentration as the x-axis (μg / mL) and the corresponding average peak area as the y-axis (mAU·s).

[0051] The raw data for the standard curve are shown in Table 1: Table 1 Linear regression yielded the regression equation A = 17.28C + 2.6, with a correlation coefficient R² = 0.9998. Here, A represents the peak area (mAU·s), and C represents the concentration of naringin in the injection solution (μg / mL).

[0052] 6. Accurately measure 5.00 mL of the sample to be tested and place it in a 25 mL volumetric flask. Add methanol to near the mark, stopper, and shake thoroughly. Place the volumetric flask in an ultrasonic cleaner and ultrasonically extract for 15 minutes at room temperature (40 kHz, 200 W). After extraction, remove the volumetric flask, cool to room temperature, and dilute to the 25 mL mark with methanol. Stopper and shake well. Filter the above solution through a 0.45 μm polytetrafluoroethylene organic phase filter membrane. Discard approximately 2 mL of the initial filtrate and collect the subsequent filtrate in a vial as the test solution. During sample pretreatment, care should be taken to avoid concentration changes due to solution evaporation.

[0053] 7. Determination and Calculation: Place the test solution in an autosampler and inject 10 μL under the chromatographic conditions described above. Record the peak area of ​​naringin. Substitute the measured peak area into the standard curve regression equation A = 17.28C + 2.6 to calculate the concentration of naringin in the test solution (μg / mL). Then, calculate the content of naringin in the original sample using the following formula: Naringin content in the original sample (g / L) = (concentration of the test solution × dilution factor) / 1000 The dilution factor is 25mL / 5.00mL = 5 times.

[0054] 8. Calculation of debittering rate before and after fermentation: Take samples of crude peel pulp before fermentation and debittered peel pulp after fermentation, respectively, and determine the naringin content according to the method described above. The debittering rate is calculated using the following formula: Naringin debittering rate (%) = (Naringin content before fermentation - Naringin content after fermentation) / Naringin content before fermentation × 100%.

[0055] 9. Precision and Accuracy Control: For each batch of samples, a standard curve midpoint (e.g., 50 μg / mL) should be measured as a quality control sample. The deviation between the measured value and the theoretical value should be within ±5%. Otherwise, the standard solution must be prepared again and the standard curve must be re-established. Each sample should be measured twice in parallel, and the average peak area should be used for calculation.

[0056] The data on the naringin content of each sample are shown in Table 2: Table 2 II. Determination of dietary fiber content; 1. Determination Principle: After homogenization, the sample is sequentially hydrolyzed using heat-resistant α-amylase, protease, and amyloglucosidase to remove starch and protein, yielding a dietary fiber hydrolysate that cannot be hydrolyzed by digestive enzymes. The hydrolysate is directly filtered and washed with hot water. The residue is collected, washed, dried, and weighed to determine the mass of insoluble dietary fiber residue. The filtrate is collected and precipitated with four times its volume of 95% ethanol preheated to 60°C, causing soluble dietary fiber to precipitate from the solution. The precipitate is dried and weighed to determine the mass of precipitable soluble dietary fiber residue. The total dietary fiber residue mass, insoluble dietary fiber residue mass, and precipitable soluble dietary fiber residue mass are all calculated by subtracting the residual protein content, ash content, and corresponding reagent blank mass from the residue. This allows for the calculation of the soluble dietary fiber content in the sample.

[0057] 2. Reagents and Materials: 95% ethanol, acetone, petroleum ether (boiling range 30℃~60℃), sodium hydroxide, concentrated hydrochloric acid, glacial acetic acid, tris(hydroxymethyl)aminomethane (Tris), maleic acid, calcium chloride dihydrate, concentrated sulfuric acid. Heat-stable α-amylase (enzyme activity ≥3000 U / mL), protease (enzyme activity ≥300 U / mL), and amylase (enzyme activity ≥2000 U / mL). MES-TRIS buffer (0.05 mol / L, pH 8.2): Weigh 19.52 g MES (2-morpholinoethanesulfonic acid) and 12.2 g Tris (tris(hydroxymethyl)aminomethane), dissolve in approximately 1.7 L of distilled water, adjust the pH to 8.2 with 6 mol / L sodium hydroxide solution (Note: adjust pH to 8.2 at 24℃; adjust pH to 8.3 at 20℃; adjust pH to 8.1 at 28℃; correct for temperature deviations using interpolation), and dilute with water to 2 L.

[0058] 3. Instruments and equipment: analytical balance (sensitivity 0.1 mg), constant temperature water bath or heating shaker (temperature control accuracy ±1℃), pH meter, crucible filter (with coarse and fine pores, model selected according to sample), vacuum filtration device, drying oven (temperature control 105℃±2℃), muffle furnace (temperature control 550℃±25℃), desiccator (containing effective desiccant).

[0059] 4. Sample Pretreatment: After thoroughly shaking the grapefruit juice sample to be tested, accurately weigh 2.000 g of the well-mixed sample (accurate to 0.001 g) and place it in a 250 mL tall beaker. If the fat content of the sample is high (>10%), it needs to be defatted with petroleum ether first: add 25 mL of petroleum ether to the beaker, shake for 5 minutes, let stand, discard the upper layer of petroleum ether, repeat twice, and evaporate the residue in a 40 °C oven until there is no petroleum ether odor. The grapefruit juice sample of this invention has a low fat content and does not require defatting treatment; it can be directly weighed and measured.

[0060] 5. Enzymatic hydrolysis: Add 40 mL of MES-TRIS buffer (pH 8.2) to a 250 mL tall beaker containing the sample, cover with aluminum foil, and shake in a 95–100 °C water bath. Once the contents of the beaker reach 95 °C, add 50 μL of heat-stable α-amylase solution (enzyme activity ≥3000 U / mL), mix gently, cover with aluminum foil, and continue shaking in a 95–100 °C water bath for 35 minutes.

[0061] Remove the beaker and cool to 60°C. Adjust the pH to 4.5 with 1 mol / L hydrochloric acid or 1 mol / L sodium hydroxide solution. Add 50 μL of protease solution (enzyme activity ≥300 U / mL), cover with aluminum foil, and shake in a 60°C water bath for 30 minutes.

[0062] Adjust the pH to 4.5 (using 1 mol / L hydrochloric acid solution or 1 mol / L sodium hydroxide solution), add 100 μL of amylase solution (enzyme activity ≥2000 U / mL), cover with aluminum foil, and shake in a 60℃ water bath for 30 minutes.

[0063] 6. Isolation and determination of soluble dietary fiber: After enzymatic hydrolysis, the hydrolysate is directly filtered through a pre-weighed crucible filter (coarse pores), and the residue is washed three times with approximately 10 mL of hot water at 60–70°C. The filtrate and washings are combined in another clean beaker. The filter residue (i.e., insoluble dietary fiber) is collected for subsequent processing (this method only measures soluble dietary fiber; the insoluble dietary fiber residue does not require further processing).

[0064] Add 95% ethanol preheated to 60°C to the collected filtrate and washings. The amount added should be four times the volume of the filtrate (to achieve a final ethanol volume fraction of 78%), while stirring slowly. After adding, cover with aluminum foil and allow to settle at room temperature for 1 hour.

[0065] The precipitate was filtered through a pre-weighed crucible filter (fine pores). The residue was washed twice with 78% ethanol (approximately 15 mL each time), twice with 95% ethanol (approximately 15 mL each time), and twice with acetone (approximately 10 mL each time). The solution was dried after each wash.

[0066] Place the crucible with residue in a 105°C drying oven and dry it to constant weight (approximately 4–6 hours). Remove it and place it in a desiccator to cool to room temperature. Weigh it and record the total mass of the residue and the crucible.

[0067] 7. Determination of Protein and Ash Content: Take the dried soluble dietary fiber residue described above and determine the protein content in the residue using the Kjeldahl method as specified in GB 5009.5 "National Food Safety Standard - Determination of Protein in Food". Take another portion of the soluble dietary fiber residue, place it in a pre-weighed crucible, and ashing it in a muffle furnace at 550℃±25℃ for 3 hours. After cooling, weigh the residue and calculate the ash content.

[0068] 8. Reagent blank determination: Following the same enzymatic hydrolysis, precipitation, filtration, washing, drying, and weighing steps, without adding a sample, perform a blank test and determine the mass of the reagent blank residue.

[0069] 9. Result Calculation: Soluble dietary fiber content is calculated using the following formula: X = (M1 - M2 - Mp- Ma - M_b) / m × 100 In the formula: X — The content of soluble dietary fiber in the sample, in grams per 100 grams (g / 100g). M1—Total mass of the crucible and soluble dietary fiber residue after drying, in grams (g). M2 — Constant weight mass of the crucible, in grams (g); Mp — the mass of protein in the residue, in grams (g). Ma — the mass of ash in the residue, in grams (g). M_b — Mass of reagent blank residue, in grams (g); m — the sample weight, in grams (g) (in this method, m = 2.000g).

[0070] Soluble dietary fiber content (g / 100mL) = X × Sample density (g / mL) / 100.

[0071] For the grapefruit juice sample, the density is approximately 1.02 g / mL, and the conversion factor is 1.02 / 100 = 0.0102.

[0072] That is: Soluble dietary fiber content (g / 100mL) = X (g / 100g) × 0.0102 × 100 = X × 1.02 / 100.

[0073] Simplified calculation: C_SDF (g / 100mL) = X × 0.0102.

[0074] The results of the determination of soluble dietary fiber content in each sample are shown in Table 3: Table 3 III. Determination of essential oil retention rate; 1. Measurement Principle: The main components of grapefruit peel essential oil (such as limonene, linalool, and other terpenoids) exhibit characteristic absorption in the ultraviolet region, with a maximum absorption wavelength of 283 nm. After extraction with anhydrous ethanol and concentration under reduced pressure, the absorbance of the sample was measured at 283 nm, and the essential oil concentration was calculated based on a standard curve. By comparing the essential oil content in samples on the day of preparation (initial) and after 6 months of storage, the essential oil retention rate was calculated, and the protective effect of gradient microencapsulation on the stability of the essential oil was evaluated.

[0075] 2. Establishing a standard curve: Accurately weigh 100.0 mg (accurate to 0.1 mg) of grapefruit peel essential oil extracted in step 1 of this invention, place it in a 100 mL volumetric flask, dissolve it with anhydrous ethanol and dilute to the mark, shake well to obtain a standard stock solution with a concentration of 1.0 mg / mL (Note: This is a standard stock solution, which is used to establish a standard curve after dilution).

[0076] Accurately measure an appropriate amount of the standard stock solution and dilute it with anhydrous ethanol to prepare a series of standard working solutions of the following concentrations, as shown in Table 4: Table 4 A standard working solution of 50 μg / mL was used, with anhydrous ethanol as a blank, and ultraviolet absorption spectroscopy was performed in the wavelength range of 200–400 nm. The results showed that grapefruit peel essential oil had a maximum absorption peak at a wavelength of 283 nm, so 283 nm was selected as the detection wavelength.

[0077] Using anhydrous ethanol as a blank, the absorbance of each standard working solution was measured at a wavelength of 283 nm. Linear regression was performed with the standard working solution concentration (C, μg / mL) as the x-axis and the absorbance (A) as the y-axis.

[0078] The standard curve equation for this experiment is: A = 0.0321C + 0.0087; correlation coefficient: R² = 0.9995; linear range: 10–200 μg / mL.

[0079] 3. Sample pretreatment: Initial sample collection: On the day the finished grapefruit juice is prepared (samples are taken immediately after bottling, sterilization, and cooling), 100.0 mL is accurately measured to determine the initial essential oil content. This value is the baseline value and serves as the basis for calculating the retention rate.

[0080] Sample collection after storage: After the same batch of finished products was stored at room temperature (20-25℃) and protected from light for 6 months, 100.0 mL was accurately measured using the same method to determine the residual essential oil content.

[0081] Place 100.0 mL of grapefruit juice sample into a 250 mL separatory funnel, add 50 mL of anhydrous ethanol, stopper the funnel, shake thoroughly for 5 minutes, allow to stand and separate into layers, and collect the ethanol phase (upper layer). Repeat the extraction once more with 30 mL of anhydrous ethanol, and combine the two ethanol extracts.

[0082] If emulsification occurs during extraction, the separatory funnel can be placed in an ultrasonic cleaner and ultrasonically treated for 5 minutes at room temperature, 40 kHz, and 200 W, then allowed to stand to separate into layers. No severe emulsification occurred during the extraction of any batches of samples using this method. This step should be performed as needed; in this experiment, all samples were directly allowed to stand to separate into layers.

[0083] The combined ethanol extract was transferred to the evaporation flask of a rotary evaporator and concentrated under reduced pressure to about 5 mL under a water bath at 40 °C and a vacuum of 0.08–0.09 MPa (the water bath temperature should be controlled not to exceed 40 °C during the concentration process to prevent the loss of low-boiling-point components in the essential oil through volatilization).

[0084] Transfer the concentrated solution to a 10 mL volumetric flask, wash the evaporation flask three times with anhydrous ethanol (about 1 mL each time), add the washing solution to the volumetric flask, dilute to the mark with anhydrous ethanol, and shake well to obtain the test solution.

[0085] Based on the essential oil concentration range determined in the preliminary experiment, the test solution was appropriately diluted (in this experiment, the test solution of each sample was directly measured or diluted 2 times) so that the absorbance fell within the linear range of the standard curve. The dilution factor was recorded.

[0086] 4. Absorbance determination: Using anhydrous ethanol as a blank, inject the test solution into a 1 cm quartz cuvette and measure the absorbance at a wavelength of 283 nm. Each sample should be measured in triplicate, and the average value should be taken.

[0087] 5. Result Calculation: The concentration of essential oil in the test solution is calculated using the following formula: Cinstrument (μg / mL) = (A - 0.0087) / 0.0321; where: A is the absorbance of the test solution at 283 nm.

[0088] The essential oil content in the original sample is calculated using the following formula: Csample (g / 100mL) = Cinstrument × Vvolume × D / Vsample / 1000 × 100; In the formula: C. Instrument – ​​Concentration of essential oil in the test solution (μg / mL); Vdilution—the volume of the test solution diluted to the final volume (mL), which is 10 mL in this method; D – Dilution factor; V sampling – Initial sampling volume (mL), which is 100 mL in this method; 1000 is the conversion factor for converting μg to mg (1000μg=1mg), then divide by 1000 to convert to g; 100 — Conversion factor for converting g / mL to g / 100mL.

[0089] Simplified calculation: Csample (g / 100mL) = Cinstrument × 10 × D / 100 / 1000 × 100 = Cinstrument × D / 1000; That is: Csample (g / 100mL) = Cinstrument × D / 1000; Essential oil retention rate (%) = C6 months / C0 × 100% In the formula: C0——The essential oil content (g / 100mL) on the day of finished product preparation; C6 months – Essential oil content (g / 100mL) after the finished product has been stored at room temperature away from light for 6 months.

[0090] The results of the essential oil retention rate determination for each sample are shown in Table 5: Table 5 IV. Sensory evaluation methods; Composition of the Evaluation Team: The sensory evaluation team consists of 10 professionally trained evaluators. All evaluators were selected from the organization's food research and development, quality control, and product testing departments. Selection criteria included: no smoking habit, no history of olfactory or gustatory disorders, and at least one year of experience in food sensory evaluation. All evaluators signed informed consent forms before formally participating in this evaluation and were informed that the evaluation purpose was solely for product quality assessment and did not involve any health risks.

[0091] Basic information of the evaluators: Of the 10 evaluators, 4 are male and 6 are female, aged between 25 and 45, with an average age of 33. All evaluators do not have olfactory or gustatory disorders, and did not consume any irritating foods (such as spicy foods, coffee, alcoholic beverages, etc.) within one hour prior to the evaluation, nor did they use cosmetics or skincare products containing fragrances. Each evaluator rinsed their mouth three times with purified water for approximately 10 seconds each time before the start of each day's evaluation to remove any residual odors.

[0092] Prior to the formal evaluation, all evaluators participated in a two-day preparatory training (approximately two hours per day). The training consisted of three parts: The first part is theoretical training, in which the host introduces the sensory characteristics of the grapefruit juice product of this invention, so that the evaluators understand the normal range of sensory attributes of grapefruit juice, including the expected color, aroma, taste and mouthfeel characteristics, as well as possible defective characteristics (such as bitterness, roughness, etc.).

[0093] The second part involved the preliminary evaluation of reference samples. The presenter prepared reference samples with known bitterness intensities and roughness grades. The bitterness reference samples included: pure water without bitterness (bitterness score 10 points), pure water containing 0.01 g / L naringin standard solution (bitterness score approximately 7 points), pure water containing 0.05 g / L naringin standard solution (bitterness score approximately 4 points), and pure water containing 0.2 g / L naringin standard solution (bitterness score approximately 1 point). The roughness reference samples included: completely smooth pure water (roughness score 10 points), a suspension containing 0.5% microcrystalline cellulose (roughness score approximately 6 points), and a suspension containing 2% microcrystalline cellulose (roughness score approximately 2 points). Each evaluator tasted and scored each of the above reference samples. The presenter verified the scoring results on-site and corrected any deviations until the deviation of each evaluator's score from the standard value did not exceed ±1.0 point.

[0094] The third part is the preliminary evaluation of the actual samples. The host provided a set of grapefruit juice samples that did not participate in the formal experiment (not the experimental samples of this invention). The evaluators scored them independently and then summarized and discussed them to unify the evaluators' understanding and grasp of the scoring criteria.

[0095] After the training, the scoring deviations of the 10 evaluators for the reference samples were all controlled within ±0.5 points, indicating that the training was up to standard and could proceed to the formal evaluation stage.

[0096] Each comparative example used the same batch of grapefruit raw materials, strains, enzyme preparations, and chemical reagents as Example 1, ensuring that all samples were completely identical under all conditions except for the single variable examined in the comparative examples. All samples (the finished products of Example 1 and Comparative Examples 1 to 5) were subjected to sensory evaluation within 48 hours after preparation to avoid the influence of differences in storage time on the evaluation results.

[0097] Sample evaluation employed a blinded coding method, meaning each sample was randomly assigned a three-digit number (e.g., "317", "852", "461", etc.) before being provided to the evaluators. Evaluators were unaware of the sample's true identity during the evaluation process (not knowing which was Example 1 and which was the comparative example). The coding was performed by a single researcher not involved in the evaluation, and evaluators were prohibited from exchanging sample number information with each other. The evaluation order of each sample was determined using a randomization table to eliminate order effects.

[0098] For each evaluation, each evaluator received a set of six samples (one each of Example 1 and Comparative Examples 1 to 5), each sample being approximately 30 mL, placed in a disposable tasting cup, with a corresponding three-digit label affixed to the cup. All samples were presented at the same temperature (approximately 25°C, room temperature), and the containers used were of the same model and specifications.

[0099] Each indicator was scored on a 10-point scale, with evaluators assigning scores in multiples of 0.5 (i.e., accurate to 0.5 points, such as 8.0, 8.5, 9.0, etc.) based on their own impressions. Each sample was scored on the three indicators in sequence: first, roughness; then, bitterness; and finally, overall taste.

[0100] Roughness rating criteria: Roughness refers to the smoothness or intensity of the grainy or fibrous feel of the sample in the mouth. 10 points indicates a completely smooth texture with no grainy or fibrous feel, like pure water or clear fruit juice; 8 to 9 points indicates a smooth texture with almost no grainy feel and no foreign body sensation when swallowing; 6 to 7 points indicates a slight grainy or fibrous feel, but it does not affect the overall drinking experience and mouthfeel comfort; 4 to 5 points indicates a noticeable grainy feel, a slightly rough texture, and the presence of suspended matter or fiber can be clearly perceived when drinking; 2 to 3 points indicates a strong fibrous feel, a rough texture, and significant discomfort when swallowing; 1 point indicates an extremely strong fibrous feel, a coarse texture, difficult to swallow, and completely unacceptable.

[0101] Bitterness rating criteria: Bitterness refers to the intensity of bitterness perceived by the tongue (especially the back of the tongue) after the sample enters the mouth. 10 points indicates no bitterness at all, consistent with the sensation of pure water; 8 to 9 points indicate almost no bitterness, only a slight bitterness detectable with very careful listening; 6 to 7 points indicate a slight bitterness, but mild and not unpleasant, within an acceptable range; 4 to 5 points indicate a noticeable bitterness, clearly perceptible when drinking, but still within acceptable limits; 2 to 3 points indicate a strong bitterness, exceeding the normal acceptable range, causing unpleasant drinking; 1 point indicates an extremely strong bitterness, making it impossible to continue drinking, causing immediate resistance upon tasting. When assessing bitterness, evaluators should hold the sample in their mouth for about 5 seconds before spitting it out or swallowing, focusing on the bitter stimulation at the back of the tongue.

[0102] Overall Taste Rating Criteria: Overall taste is a holistic indicator that reflects the evaluator's comprehensive assessment of the sample's overall flavor, mouthfeel, and drinking pleasure, rather than a score for a single characteristic. 10 points indicates very satisfied; the sample excels in aroma, taste, smoothness, and harmony, and the evaluator is willing to drink it and enjoy tasting it again. 8-9 points indicates satisfied; the sample has a good overall taste, with possible minor flaws that do not affect the overall pleasure. 6-7 points indicates basically satisfied; the sample has slight shortcomings in one or more aspects, but is still acceptable overall and meets the standards of a qualified product. 4-5 points indicates somewhat dissatisfied; the sample has obvious defects (such as excessive bitterness, overly rough texture, or unharmonious flavors), and although still drinkable, the experience is unpleasant. 2-3 points indicates very dissatisfied; the sample's defects are very prominent and unacceptable. 1 point indicates completely unacceptable; the sample lacks the basic qualities required for consumption as a beverage. When evaluating the overall taste, evaluators need to consider multiple dimensions, including aroma intensity, aroma harmony, sweetness, acidity, balance of bitterness, smoothness, and aftertaste.

[0103] The evaluation was conducted in a quiet, odorless, and well-lit independent sensory evaluation room, with the room temperature controlled between 22°C and 25°C. Each evaluator sat alone at an evaluation table, which were separated by partitions to avoid mutual interference.

[0104] Before the evaluation begins, the host gives the evaluators an evaluation instruction, reminding them to evaluate the samples in the order of their blind sample numbers, rinse their mouths with purified water before evaluating each sample, and allow at least 60 seconds between two samples to allow their mouths to return to a normal state.

[0105] Each evaluator independently scores all samples based on the three indicators. During the evaluation process, they are not allowed to discuss the scoring content with other evaluators or inquire about the scoring results of others. The total evaluation time for each sample should be controlled within 3 to 5 minutes to avoid the influence of fatigue effect on judgment.

[0106] After all evaluators have completed their scoring, the host collects the scoring sheets, checks whether each sheet is filled out completely (whether any samples or indicators have been missed), and enters the data after confirming that everything is correct.

[0107] For each indicator of each sample, the arithmetic mean of the scores from 10 evaluators was calculated, rounded to one decimal place. The standard deviation was also calculated to assess the dispersion of the data and the consistency among evaluators. For any indicator, if an evaluator's score deviated from the average of the other nine evaluators by more than ±2.0 points, the outlier was removed and the average was recalculated (no such outliers occurred in this experiment). The final average was used as the sensory score for that indicator.

[0108] One-way ANOVA was used to test the significance of differences in sensory scores among samples, with P < 0.05 considered statistically significant. When the ANOVA results were significant, Tukey's HSD post-hoc test was used to perform pairwise comparisons between samples to determine which samples had significant differences.

[0109] The following are the original scores from 10 evaluators for the three sensory indicators of each sample (Example 1 and Comparative Examples 1 to 5). Evaluators are numbered E01 to E10.

[0110] The raw data for roughness scoring are shown in Table 6: Table 6 The average and standard deviation of the roughness scores for each sample are shown in Table 7. Table 7 The raw data for bitterness scores are shown in Table 8: Table 8 The mean and standard deviation of the bitterness scores for each sample are shown in Table 9: Table 9 The raw data for the overall taste score are shown in Table 10: Table 10 The average and standard deviation of the overall taste scores for each sample are shown in Table 11: Table 11 The sensory evaluation results of each sample are summarized in Table 12: Table 12 Data Result Analysis: Compared to Example 1, Comparative Example 1 showed a significant decrease in soluble dietary fiber content from 2.8 g / 100 mL to 0.2 g / 100 mL (a reduction of 92.9%), indicating that the dietary fiber in the peel was the core source of nutrition for the whole fruit in Example 1. The debittering rate data for naringin and limonene were not applicable (fermentation was not involved); the 6-month essential oil retention rate data were also not applicable (no essential oils were added). The texture roughness score slightly increased from 8.6 to 9.2 (only the pulp became more delicate), and the bitterness score decreased from 1.0 to 0.5 (the pulp was almost entirely free of bitterness). However, the overall taste score dropped significantly from 8.6 to 6.8 (a reduction of 20.9%) because the product lacked the characteristic grapefruit flavor from the peel (the full-bodied taste from essential oils and dietary fiber), resulting in a single aroma and significantly reduced nutritional value. The comparative example confirms that although the whole fruit utilization feature is not inferior to Example 1 in terms of individual indicators such as "roughness" and "bitterness", the overall taste (especially the fullness of flavor and nutritional value) is significantly deteriorated. This indicates that the dietary fiber and essential oil components in the peel make an indispensable contribution to the overall quality of the product. Whole fruit utilization is the core foundation for achieving a balance between nutrition and taste in this invention.

[0111] Compared to Example 1, Comparative Example 2 showed that the naringin content increased from 0.014 g / L to 0.153 g / L (an increase of 992.9%), the debittering rate of naringin decreased significantly from 96.6% to 77.5% (a decrease of 19.8%), and the debittering rate of limonene decreased sharply from 94.2% to 42.1% (a decrease of 55.3%), indicating that *Lactobacillus plantarum* plays an irreplaceable role in the degradation of limonene-like bitter substances compared to *Aspergillus niger*. The soluble dietary fiber content decreased slightly from 2.8 g / 100 mL to 2.7 g / 100 mL (essentially unchanged, indicating that enzymatic modification still played a role), and the 6-month essential oil retention rate decreased slightly from 80.5% to 79.8% (essentially unchanged, indicating that microencapsulation still played a role). The bitterness score increased sharply from 1.0 to 3.8 (an increase of 280%), and the overall taste score decreased from 8.6 to 5.8 (a decrease of 32.6%). This comparative study confirms that the Lactobacillus plantarum component in the compound strain (Aspergillus niger + Lactobacillus plantarum) plays a key role in the removal of limonene-like bitter substances. Aspergillus niger alone cannot effectively remove the bitterness of limonene. The synergistic effect of the compound strain is the core guarantee for achieving the "extremely low bitterness" technical effect.

[0112] Compared to Example 1, Comparative Example 3 showed a significant decrease in soluble dietary fiber content from 2.8 g / 100 mL to 0.9 g / 100 mL (a decrease of 67.9%), indicating that enzymatic modification is a key step in converting insoluble dietary fiber into soluble dietary fiber. Naringin content slightly increased from 0.014 g / L to 0.015 g / L (essentially unchanged), the debittering rate remained at 96.5% (fermentation steps were unaffected), the limonene debittering rate was 93.8% (essentially unchanged), and the 6-month essential oil retention rate slightly decreased from 80.5% to 80.2% (essentially unchanged). The texture roughness score significantly decreased from 8.6 to 4.8 (a decrease of 44.2%), and the overall texture score decreased from 8.6 to 4.8 (a decrease of 44.2%). This comparative study confirms that enzymatic modification is a crucial step in transforming the rough, insoluble dietary fiber in fruit peel into smooth, soluble dietary fiber. Without enzymatic modification, the dietary fiber in the peel retains its rough texture, directly resulting in a coarse and unpalatable product taste. Furthermore, enzymatic modification serves as a follow-up step after fermentation-based debittering, and its conversion efficiency depends on the initial softening of the peel tissue during fermentation, further validating the importance of the inter-step process.

[0113] Compared to Example 1, the essential oil retention rate in Comparative Example 4 decreased sharply from 80.5% to 35.2% after 6 months (a decrease of 56.3%), indicating that the essential oils that were not microencapsulated rapidly volatilized and oxidized during storage. The naringin content was 0.014 g / L (the same as in Example 1), the debittering rate was 96.6% (the same), the limonene debittering rate was 94.0% (the same), and the soluble dietary fiber content was 2.8 g / 100 mL (the same), indicating that omitting microencapsulation had no effect on other indicators. The roughness score decreased slightly from 8.6 to 8.4 (basically the same), and the bitterness score was 1.0 (the same), but the overall taste score decreased significantly from 8.6 to 5.6 (a decrease of 34.9%). This is because the direct addition of the essential oil resulted in an overly stimulating (unharmonious) aroma in the initial stage, and after storage, a large amount of aroma dissipated, resulting in a bland flavor when consumed. This comparative study confirms that gradient microencapsulation is a key technology for protecting essential oils from volatilization and oxidation, achieving long-lasting sustained release of aroma components and product flavor stability.

[0114] Compared to Example 1, Comparative Example 5 showed that the naringin content increased from 0.014 g / L to 0.118 g / L (an increase of 742.9%), the debittering rate of naringin decreased from 96.6% to 82.1% (a decrease of 15.0%), and the debittering rate of limonene decreased from 94.2% to 72.5% (a decrease of 23.0%), indicating that the fermentation debittering efficiency was significantly reduced after reversing the order of steps. The soluble dietary fiber content decreased from 2.8 g / 100 mL to 2.1 g / 100 mL (a decrease of 25.0%) because the soluble sugars released during the initial enzymatic modification were consumed by the microbial metabolism in the subsequent fermentation, altering the fermentation metabolic environment. The texture roughness score decreased from 8.6 to 6.5 (a decrease of 24.4%), the bitterness score increased from 1.0 to 2.8 (an increase of 180%), and the overall texture score decreased from 8.6 to 5.2 (a decrease of 39.5%). This comparative example confirms that the order of steps 2 (fermentation) and 3 (enzymatic hydrolysis) in this invention has a specific scientific basis—the sequential arrangement of fermentation followed by enzymatic hydrolysis ensures that the microorganisms preferentially utilize the bitter substances in the fruit peel as metabolic substrates, while the soluble sugars produced by enzymatic hydrolysis are not consumed by fermentation and can be completely retained in the product; reversing the order causes the fermenting microorganisms to preferentially consume the sugars released by enzymatic hydrolysis, resulting in decreased debittering efficiency, insufficient sweetness in the product, and an unbalanced flavor. The specificity of the step order is a key guarantee for the seamless coordination of each process.

[0115] The above description is merely an embodiment of the present invention and does not limit the patent scope of the present invention. Any equivalent modifications made based on the content of the present invention specification, or direct or indirect applications in related technical fields, are similarly included within the patent protection scope of the present invention.

Claims

1. A method for preparing grapefruit juice based on whole-fruit debittering microencapsulation, characterized in that, Includes the following steps: Step 1: Pre-treatment of whole pomelo: Separate the pomelo peel from the pulp. Collect the oil cell layer and white peel layer of the peel separately. Remove the seeds from the pulp. Extract pomelo peel essential oil from the oil cell layer peel. Combine the extracted oil cell layer residue with the white peel layer peel, cut into pieces, add water and blend into a pulp to obtain a coarse peel pulp. Add water to the pulp and blend into a pulp to obtain a coarse pulp pulp. Step 2: Debittering by microbial directional fermentation of fruit peel: Mix Aspergillus niger and Lactobacillus plantarum at a live cell ratio of 1:1 to 3:1 to prepare a compound fermentation inoculum. Add the compound fermentation inoculum to the crude fruit peel pulp obtained in Step 1. The inoculation amount is 2% to 6% of the mass of the crude fruit peel pulp. Perform anaerobic-aerobic two-stage fermentation at 28 to 32°C. After fermentation, sterilize and cool to obtain debittered fruit peel pulp. Step 3: Enzymatic hydrolysis and synergistic modification of fruit peel dietary fiber: A compound enzyme preparation, consisting of cellulase, pectinase and xylanase, is added to the debittered fruit peel pulp obtained in Step 2. The enzymatic hydrolysis reaction is carried out at 45-55℃ and pH 4.5-5.5 for 60-180 minutes. Ultrasonic treatment is used during the enzymatic hydrolysis process. After the enzymatic hydrolysis is completed, the enzyme is inactivated and cooled to obtain the modified fruit peel pulp. Step 4: Enzymatic hydrolysis and clarification of fruit pulp: Add pectinase to the crude fruit pulp obtained in Step 1, and hydrolyze for 30 to 90 minutes at 45-50℃ and pH 3.5-4.

5. After enzyme inactivation, centrifuge and collect the supernatant to obtain clarified fruit pulp juice. Step 5: Gradient microencapsulation of grapefruit peel essential oil: The grapefruit peel essential oil obtained in Step 1 is subjected to primary microencapsulation and secondary microencapsulation in sequence. The primary microencapsulation uses β-cyclodextrin as the wall material for encapsulation, and the secondary microencapsulation uses sodium alginate-calcium chloride system for solidification and encapsulation, to obtain gradient microencapsulated essential oil microcapsules. Step 6: Whole-fruit grapefruit juice preparation and microencapsulation integration: Mix the modified peel pulp obtained in Step 3 and the clarified pulp juice obtained in Step 4, add the gradient microencapsulated essential oil microcapsules obtained in Step 5, homogenize, deaerate, sterilize, and fill to obtain the finished grapefruit juice based on whole-fruit debittering microencapsulation.

2. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, In step 1, the peel is cut into pieces and water is added at a ratio of 1:2 to 1:5, and then pulped at 40 to 50°C; the pulp is pulped at a ratio of 1:1 to 1:

3.

3. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, The total viable cell concentration of the compound fermentation inoculum in step 2 is 10. 8 ~10 9 CFU / mL; the two-stage fermentation consists of anaerobic fermentation for 12-24 hours followed by aerobic fermentation with sterile air for 6-12 hours, for a total fermentation time of 18-36 hours.

4. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, In step 2, 0.5% to 2.0% of the mass of the fruit peel pulp is added to the fruit peel pulp as a carbon source, wherein the carbon source is one or more of glucose, sucrose or fructooligosaccharides; the fermentation pH is 5.0 to 5.

5.

5. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, In step 3, the enzyme activity ratio of cellulase, pectinase and xylanase in the compound enzyme preparation is 2:1:1, and the total amount of compound enzyme preparation added is 0.1% to 0.5% of the mass of the debittered fruit peel pulp.

6. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, The ultrasonic treatment in step 3 has a power of 200-400W and is performed intermittently, with a 2-5 minute interval between ultrasonic treatments of 5-10 minutes.

7. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, The specific method for primary microencapsulation described in step 5 is as follows: β-cyclodextrin is dissolved in water at 40-50°C, grapefruit peel essential oil is added under stirring conditions, and the mass ratio of essential oil to β-cyclodextrin is 1:8 to 1:

15. The mixture is stirred and encapsulated at 50-60°C for 1-2 hours, cooled, allowed to stand, filtered, washed, and dried to obtain primary essential oil microcapsules.

8. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, The specific method for secondary microencapsulation in step 5 is as follows: the primary essential oil microcapsules are dispersed in a sodium alginate solution with a mass fraction of 1% to 3%, and then dropped into a calcium chloride solution with a mass fraction of 2% to 5% using the sharp-pore coagulation bath method to solidify for 30 to 60 minutes. After filtration, washing, and drying, gradient microencapsulated essential oil microcapsules are obtained.

9. The method for preparing grapefruit juice based on whole-fruit debittering microencapsulation according to claim 1, characterized in that, In step 6, the mixing mass ratio of the modified peel pulp to the clarified pulp juice is 1:3 to 2:3; the amount of gradient microencapsulated essential oil microcapsules added is 0.5% to 2.0% of the mass of the whole grapefruit juice base; the homogenization pressure is 20 to 25 MPa, and the homogenization is performed 1 to 2 times.

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