Low-bitterness pomelo whole fruit powder for improving intestinal microecological disorder and preparation method thereof

Abstract

This invention discloses a method for preparing low-bitterness Shatin pomelo whole fruit powder that improves intestinal microecological disorder, comprising: Step 1: removing the oil cell layer from the Shatin pomelo, cutting it into small pieces and removing the seeds, adding distilled water, and pulping to obtain a free-flowing Shatin pomelo homogenate A; Step 2: subjecting Shatin pomelo homogenate A to pulsed electric field treatment to obtain pulsed electric field pretreated Shatin pomelo homogenate B; Step 3: taking Shatin pomelo homogenate B, adding pectinase and cellulase, and stirring in a water bath; after the reaction, inactivating the enzymes to obtain Shatin pomelo homogenate C after pectinase and cellulase hydrolysis; Step 4: adding naringinase to Shatin pomelo homogenate C, and stirring in a water bath; after the reaction, inactivating the enzymes to obtain Shatin pomelo homogenate D after naringinase hydrolysis; Step 5: subjecting Shatin pomelo homogenate D to vacuum freeze-drying, then pulverizing and sieving to obtain Shatin pomelo whole fruit powder. This invention also discloses a Shatin pomelo whole fruit powder prepared by the above method and its application.

Description

Technical Field

[0001] This invention relates to a low-bitterness Shatang pomelo whole fruit powder for improving intestinal microecological disorders and its preparation method. Specifically, it relates to a method for preparing Shatang pomelo whole fruit powder based on pulsed electric field pretreatment and synergistic enzymatic debittering by pectinase, cellulase and naringinase coupling, and the application of the obtained fruit powder in health foods for improving intestinal microecological disorders. Background Technology

[0002] Shatang pomelo is a distinctive citrus fruit from South my country, holding a particularly important position in the agricultural economy of Meizhou City, Guangdong Province, and is a geographical indication fruit of Meizhou City. The Shatang pomelo fruit consists of the following layers from the outside in: oil cells, white peel, pericarp, pulp, and seeds. The peel (oil cells and white peel) and pericarp account for approximately 50% of the total fruit weight. Modern research shows that all parts of the Shatang pomelo are rich in flavonoids and dietary fiber (DF), both of which exhibit effects such as regulating glucose and lipid metabolism and improving intestinal microecological disorders. The pericarp has the highest total flavonoid and DF content, followed by the white peel. Therefore, processing the entire fruit into powder using the white peel, pericarp, and pulp is a processing method that can fully enrich the active ingredients of the pomelo. However, the white peel and pericarp, due to their high content of flavonoid glycosides, have a severely bitter taste, and their high fiber content results in a coarse texture, often leading to their disposal as waste, resulting in resource waste and nutrient loss. Therefore, using green processing methods to prepare Shatang pomelo whole fruit powder with low bitterness and rich active ingredients is of great significance for increasing the added value of Shatang pomelo and extending its industrial chain.

[0003] Currently, the main methods for debittering citrus fruit products are physical adsorption and naringinase enzymatic hydrolysis. The former primarily uses adsorbents to remove bitter flavonoid glycosides, resulting in significant loss of active ingredients; the latter utilizes naringinase to hydrolyze bitter flavonoid glycosides into non-bitter flavonoid aglycones, thus achieving debittering. However, both methods are limited to debittering liquid matrices such as fruit juice and wine. Shatang pomelo whole fruit homogenate is rich in DF (diethyltoluene), has poor fluidity, and the homogenate matrix hinders the contact between bitter substances and adsorbents or naringinase; therefore, the above methods cannot be directly used for debittering Shatang pomelo whole fruit homogenate or Shatang pomelo fruit powder. Furthermore, existing technologies rarely offer citrus whole fruit powder processing methods that simultaneously achieve efficient debittering and enrichment of active components. For example, invention patent CN113925146A discloses a method for producing debittered ultrafine grapefruit powder. The main process is as follows: whole fruit is cut into pieces, sodium salt / thickener is added and pulped, enzymes are inactivated by ultrasound and microwave, and then enzymatically hydrolyzed using a combination of enzymes such as naringinase and cellobiase. The hydrolysate is freeze-dried, soaked in ethanol, and the precipitate and supernatant are separated. The supernatant is filtered through cholesterol-ethanol precipitation, and the filtrate is freeze-dried with the precipitate soaked in ethanol and then ultrafinely pulverized to obtain debittered ultrafine grapefruit powder. Although this process uses a combination of enzymatic hydrolysis, the entire enzymatic hydrolysis system is extremely viscous, which is not conducive to the contact between naringinase and the substrate. The enzymatic debittering reaction is difficult to carry out. Debittering mainly relies on the subsequent cholesterol-ethanol precipitation step, that is, the bitter substances such as naringin soaked in ethanol react with cholesterol to form insoluble substances, and then the precipitate is filtered to remove the bitter substances. Therefore, the fruit powder prepared by this method contains almost no naringin (the removal rate is over 90%), and the flavonoid active ingredients are almost completely lost.

[0004] Against this backdrop, there is an urgent need to explore a simple and efficient whole-fruit powder debittering technology that takes into account "whole fruit utilization", "effective debittering" and "preservation of active ingredients". Summary of the Invention

[0005] One objective of this invention is to provide a method for preparing low-bitterness pomelos whole fruit powder that improves intestinal microecological disorders, a method that simultaneously reduces bitterness and modifies and retains active components; another objective is to provide pomelos whole fruit powder prepared by the above method.

[0006] One of the objectives of this invention can be achieved through the following technical solution: a method for preparing low-bitterness Shatin pomelo whole fruit powder to improve intestinal microecological disorder, comprising the following steps:

[0007] Step 1: Remove the oil cells from the pomelo, cut it into small pieces and remove the seeds to obtain the raw pomelo material; add distilled water to it and blend it to obtain a smooth pomelo homogenate A.

[0008] Step 2: Take the pomelos homogenate A obtained in Step 1 and subject it to pulsed electric field treatment to obtain pomelos homogenate B pretreated by pulsed electric field.

[0009] Step 3: Take the pomelo homogenate B obtained in Step 2, add pectinase and cellulase, and stir the reaction in a water bath; after the reaction is complete, inactivate the enzymes to obtain pomelo homogenate C after enzymatic hydrolysis by pectinase and cellulase.

[0010] Step 4: Take the pomelo homogenate C obtained in Step 3, add naringinase, and stir the reaction in a water bath; after the reaction is complete, inactivate the enzyme to obtain pomelo homogenate D after naringinase hydrolysis.

[0011] Step 5: Vacuum freeze-dry the pomelo homogenate D obtained in Step 4, then pulverize and sieve it to obtain pomelo whole fruit powder.

[0012] In step one, the tissues used to prepare the Shatin pomelo homogenate include the white peel, the pericarp, and the pulp, meaning that this invention essentially achieves full utilization of the Shatin pomelo. Preferably, distilled water is added to the Shatin pomelo raw material at a material-to-liquid ratio of 1:3 to 1:5 (w / v, g / mL) for homogenization to obtain a Shatin pomelo homogenate A with good flowability.

[0013] Preferably, in step two, the pomelo homogenate A is subjected to pulsed electric field treatment for 3 to 5 minutes under the conditions of electric field strength of 8 to 12 kV / cm and pulse frequency of 300 to 500 Hz to obtain pomelo homogenate B pretreated by pulsed electric field.

[0014] Preferably, in step three, the pH of the Shatin pomelo homogenate B is adjusted to 4.0-5.0 with citric acid solution, and 0.1-0.2% (g / g fresh weight of Shatin pomelo raw material) of pectinase and cellulase complex enzyme preparation is added. The mixture is stirred and reacted in a water bath at 45-55 ℃ for 2-4 hours, and then the enzymes are inactivated to obtain Shatin pomelo homogenate C after enzymatic hydrolysis by pectinase and cellulase; wherein the weight ratio of pectinase to cellulase is 1:1.

[0015] Preferably, in step four, the pH of the Shatin pomelo homogenate C is adjusted to 4.0-5.0 with sodium citrate solution, 0.3-0.4% (g / g fresh weight of Shatin pomelo raw material) of naringinase is added, and the mixture is stirred and reacted in a water bath at 45-55 ℃ for 3-4 h. Then the enzyme is inactivated to obtain Shatin pomelo homogenate D after naringinase hydrolysis.

[0016] Preferably, in step five, the Shatin pomelo homogenate D is evenly spread in a container with a thickness of 1~1.5 cm, placed in a freeze dryer for vacuum freeze drying until the moisture content is below 5%, and then pulverized and passed through a 40-mesh sieve to obtain Shatin pomelo whole fruit powder.

[0017] The second objective of this invention can be achieved through the following technical solution: a low-bitterness pomelos whole fruit powder that improves intestinal microecological disorder, prepared by the above preparation method.

[0018] The whole fruit powder of Shatin pomelo can be used to prepare food or health products that improve intestinal microecological disorders.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] (1) This invention prepares low-bitterness pomelo whole fruit powder that improves intestinal microecological disorder by constructing a pulsed electric field pretreatment combined with pectinase and cellulase coupled with naringinase stepwise enzymatic hydrolysis method; specifically, firstly, pulsed electric field pretreatment is used to enhance cell membrane permeability by utilizing the electroporation effect, promoting the release of intracellular substances, and providing more enzymatic hydrolysis sites for subsequent series of enzymatic hydrolysis reactions; then, pectinase and cellulase are used to efficiently destroy the cell wall network structure, converting insoluble fiber (IDF) into soluble fiber (SDF), greatly improving the juice yield. Further promote the release and dissolution of bitter flavonoid glycosides; finally, use naringinase to enzymatically hydrolyze the large amount of dissolved flavonoid glycosides, converting bitter flavonoid glycosides into non-bitter flavonoid aglycones, forming a highly efficient action chain of "physical cell wall breaking - enzymatic hydrolysis and liquefaction - specific debittering"; this action chain removes bitterness while retaining active flavonoid components, and at the same time converts IDF in fruit powder into SDF that is more easily utilized by the body. The whole process is simple and easy to operate, the reaction conditions are mild, and the exogenous chemical additives are greatly reduced, which can prepare high-purity fruit powder with preserved active ingredients and good flavor.

[0021] (2) In this invention, after treatment with a pulsed electric field synergistic with pectinase and cellulase, the juice yield of Shatang pomelo homogenate increased from 49.7-53.0% to 80.3-84.8% without treatment, the soluble pectin content increased from 2.4-2.6 mg Gal / g FW to 5.2-5.5 mg Gal / g FW, and the total flavonoid release rate increased from 47.0-50.2% to 80.5-84.3%.

[0022] (3) The whole fruit powder of Shatin pomelo prepared by the present invention has a debittering rate of 86.4-88.4%, and the taste is reduced from "relatively bitter" to "slightly bitter". Its total flavonoid content is 17.4-18.5 mg CE / g DW, which is still at a high level. The total dietary fiber (TDF) content is 47.8-48.6%. Compared with the whole fruit powder of Shatin pomelo prepared by direct drying and pulverization, the SDF content of the whole fruit powder of Shatin pomelo prepared by the present invention is increased from 7.6-8.3% to 16.2-17.0%, and the ratio of IDF to SDF is balanced (1.9-2.0).

[0023] (4) The soluble dietary fiber, especially soluble pectin, content in the whole pomelo powder prepared by this invention is significantly increased, which helps the intestinal flora to ferment slowly, selectively promotes the proliferation of beneficial intestinal bacteria and inhibits the proliferation of harmful intestinal bacteria, thereby regulating the intestinal microecological balance. Compared with whole pomelo powder prepared by direct drying and pulverization or whole pomelo powder hydrolyzed by pectinase-cellulase-naringinase, the whole pomelo powder prepared by this invention more effectively promotes the production of short-chain fatty acids (SCFAs) by fecal flora, especially butyric acid, and more effectively inhibits the production of lipopolysaccharides (LPS) by fecal flora.

[0024] (5) The whole fruit powder of Shatang pomelo prepared by the present invention has low bitterness and is rich in flavonoids and SDF, and can be used as raw material for health food or health care products to regulate intestinal microecological disorders. Attached Figure Description

[0025] Figure 1 The response surface 3D plot shows the interactive effects of naringinase addition, hydrolysis temperature, and hydrolysis time on the total degradation rate of major bitter flavonoids.

[0026] Figure 2 The SCFAs content in the fermentation broth of PEF-PCNE-SWFP prepared in Example 4, SWFP prepared in Comparative Example 1, and PCNE-SWFP prepared in Comparative Example 4 after 24 h of fermentation by fecal microbiota. When the letters on the column are completely different, it indicates a statistically significant difference (p < 0.05), the same applies below.

[0027] Figure 3 The LPS content in the fermentation broth of PEF-PCNE-SWFP prepared in Example 4, SWFP prepared in Comparative Example 1, and PCNE-SWFP prepared in Comparative Example 4 after 24 hours of fermentation by fecal microbiota. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention. The Shatang pomelos selected in the embodiments are those harvested mainly between December and January and have a high degree of maturity.

[0029] The physicochemical index testing methods for the whole fruit powder of Shatin pomelo in the following examples and comparative examples are as follows:

[0030] (1) Bitterness evaluation: Quinine was used as the bitterness standard substance, and solutions of different concentrations were prepared. The bitterness was divided into five levels: 0.5 (no bitterness), 1.0 (slightly bitter), 2.5 (slightly bitter), 5.0 (quite bitter), and 10 (extremely bitter). Whole fruit powder of Shatin pomelo was taken and distilled water was added at a material-to-liquid ratio of 1:20 (w / v). The solution was homogenized at 5000 r / min for 10 min to obtain a homogeneous solution for later use. The sensory evaluation group consisted of 20 adults aged 20-30 with normal taste. After rinsing their mouths with distilled water, the evaluators took 3 mL of quinine solution in their mouths, let it reach the back of their tongues and stay for 10 s before spitting it out to determine the taste sensation corresponding to different bitterness values. The same method was used to evaluate the bitterness of the sample solutions in turn to determine their bitterness values ​​and bitterness levels. The bitterness removal rate (%) of Shatin pomelo whole fruit powder = (A1-A2) / A1×100%, where A1 is the bitterness value of SWFP prepared in Comparative Example 1, and A2 is the bitterness value of PEF-PCNE-SWFP prepared in any of Examples 1 to 4, or NE-SWFP prepared in Comparative Example 2, or PEF-NE-SWFP prepared in Comparative Example 3, or PCNE-SWFP prepared in Comparative Example 4.

[0031] (2) Juice yield determination: Take appropriate amounts of the original pomelo homogenate prepared by direct addition of water and homogenization and the homogenate after pretreatment with pulsed electric field coupled with pectinase-cellulase enzymatic hydrolysis, respectively, centrifuge at 8000 rpm for 10 min, collect the supernatant and weigh it, and calculate the juice yield as the percentage of the weight of the supernatant to the weight of the homogenate before centrifugation.

[0032] (3) Determination of soluble pectin content: The soluble pectin content in Shatin pomelo homogenate was determined by the carbazole method. Appropriate amounts of the original Shatin pomelo homogenate and the homogenate after pulsed electric field pretreatment coupled with pectinase-cellulase hydrolysis were taken and centrifuged at 8000 rpm for 10 min, and the supernatant was collected. Four volumes of 95% ethanol were added to the supernatant, and after standing at room temperature for 1 h, the mixture was centrifuged again, and the precipitate was collected. The precipitate was then washed twice with 60% ethanol to obtain the pectin precipitate. The obtained pectin precipitate was dissolved in distilled water, and concentrated sulfuric acid and carbazole reagent were added. After mixing, the mixture was incubated at 85 ℃ for 10 min, cooled to room temperature, and the absorbance was measured at a wavelength of 525 nm. A standard curve was prepared using galacturonic acid as a standard, and the soluble pectin content in the sample was calculated. The results were expressed as mg galacturonic acid equivalent per g of fresh weight of homogenate (mg Gal / g FW).

[0033] (4) Extraction of flavonoids: For the extraction of flavonoids from the supernatant of Shatang pomelo homogenate, the supernatant of the homogenate was extracted 5 times with ethyl acetate (1:10, v / v), the extracts were combined, and the mixture was rotary evaporated to dryness at 45 °C under vacuum. The extract was dissolved in 85% methanol aqueous solution (v / v) and the volume was adjusted to 5 mL to obtain the flavonoid extract. The extract was then aliquoted and stored at -20 °C for later use. For the extraction of flavonoids from Shatang pomelo homogenate and whole fruit powder, 80% acetone solution pre-cooled at 4 °C was added at a material-to-liquid ratio of 1:4 (w / v). The mixture was homogenized at 10000 r / min for 5 min under ice bath, centrifuged, and the supernatant was collected. The residue was extracted once more in the same manner. The two extracts were combined, and the mixture was rotary evaporated to dryness at 45 °C under vacuum. The extract was dissolved in 85% methanol aqueous solution (v / v) and the volume was adjusted to 5 mL to obtain the flavonoid extract. The extract was then aliquoted and stored at -20 °C for later use.

[0034] (5) Determination of total flavonoid content: Take 1 mL of the above flavonoid extract diluted appropriately, evaporate to dryness at 45 °C by rotary evaporation, add 1 mL of tetrahydrofuran / ethanol (1:1, v / v) solution to reconstitute, transfer to a 25 mL graduated test tube, add NaBH4 solution (0.5 mL, 50 mmol / L) and AlCl3 solution (0.5 mL, 74.6 mmol / L) respectively, shake at room temperature for 30 min, add 0.5 mL of 50 mmol / L NaBH4 solution, shake at room temperature for 30 min, add 2 mL of 0.8 mol / L acetic acid solution pre-cooled at 4 °C, shake at room temperature in the dark for 15 min, add 1 mL of 20 mmol / L tetrachloroquinone solution, heat in an oil bath at 95 °C for 60 min. After the reaction, the test tube was immersed in tap water to cool. The volume of the reaction solution was brought up to 4 mL with methanol. 1 mL of 16% (m / v) vanillin methanol solution was added, and after mixing, 2 mL of 12 mol / L HCl solution was added and mixed. The mixture was then incubated at room temperature in the dark for 15 min. 200 μL of the supernatant was transferred to a 96-well plate, and the absorbance was measured at 490 nm. A standard curve was prepared using catechins as a standard. The total flavonoid content of the sample was expressed as mg catechin equivalents (CE) per g of sample, i.e., mg CE / g DW. The release rate of total flavonoids (%) = (C s / C h )×100%, where C s The total flavonoid content in the supernatant of Shatin pomelo homogenate is C. h The total flavonoid content is given in the corresponding Shatin pomelo homogenate.

[0035] (6) Determination of SDF, IDF and TDF content: A certain amount of whole pomelo powder was weighed and added to 95% ethanol solution (1:4, w / v). The mixture was soaked at room temperature for 24 h to remove small molecule sugars and pigments. After centrifugation, the supernatant was discarded. The residue was extracted twice with 95 ℃ distilled water (1:20, w / v), 2 h each time, and centrifuged. The supernatant and precipitate obtained were used for the preparation of SDF and IDF, respectively. The precipitate was washed three times with hot water at 70 ℃ and dried in an oven at 60 ℃ for 48 h to obtain IDF. The combined supernatant was concentrated to 1 / 5 of the initial volume under vacuum at 50 ℃. Four times the volume of 95% ethanol was added, and the mixture was allowed to stand at 4 ℃ for 24 h. After centrifugation, a flocculent precipitate was obtained. The flocculent precipitate was washed three times with 95% ethanol and dried in an oven at 60 ℃ for 48 h to obtain SDF. The total DF content was the sum of the contents of the two.

[0036] (7) Determination of the degradation rate of the main monomer flavonoids:

[0037] The changes in the content of major bitter flavonoids (naringin, melitidin, and cigranoside B) in Shatin pomelo homogenate before and after enzymatic hydrolysis with naringinase were determined by high performance liquid chromatography (HPLC). The chromatographic conditions were: C 18 The chromatographic column (Zorbox SB, 250 × 4.6 mm, 5 μm) was used. Mobile phase A was 0.4% acetic acid aqueous solution, and mobile phase B was acetonitrile. The gradient elution program was: 0–40 min, 5–25% B; 40–45 min, 25–35% B; 45–50 min, 35–50% B. The detection wavelength was 280 nm, the flow rate was 1.0 mL / min, the column temperature was 37 ℃, and the injection volume was 10 μL. The degradation rate (%) of monomeric flavonoids was calculated as [(C0 - C1) / C0] × 100%, where C0 and C1 were the contents of each monomeric flavonoid in the homogenate before and after naringinase hydrolysis, respectively. The total degradation rate was calculated based on the individual degradation rates of the three components and their proportion in the Shatin pomelo raw material.

[0038] Example 1:

[0039] After removing the oil cell layer and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatang pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatang pomelo homogenate A. A pulsed electric field was applied at 12 kV / cm for 3 min to obtain pulsed electric field pretreated Shatang pomelo homogenate B. The pH of the pulsed electric field pretreated homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.1% (g / g fresh weight of Shatang pomelo raw material) of a pectinase and cellulase (1:1) composite enzyme preparation was added. After stirring in a magnetic water bath at 50 ℃ for 3 h, the enzymes were inactivated at 85 ℃ for 10 min to obtain pulsed electric field pretreated Shatang pomelo homogenate C coupled with pectinase-cellulase enzymatic hydrolysis. After cooling, the pH was adjusted to 4.5 with 1 M sodium citrate solution. 0.4% (g / g fresh weight of Shatang pomelo raw material) of naringinase was added, and enzymatic hydrolysis was continued at 50 ℃ for 3 h. Then, the enzyme was inactivated at 85 ℃ for 10 min to obtain Shatang pomelo homogenate D after pulsed electric field pretreatment coupled with pectinase-cellulase-naringinase enzymatic hydrolysis. Shatang pomelo homogenate D was evenly spread in a container to a thickness of 1.5 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain low-bitterness Shatang pomelo whole fruit powder enriched with flavonoids and SDF.

[0040] Example 2:

[0041] After removing the oil cell layer and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatang pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:3 (w / v), and homogenized to obtain Shatang pomelo homogenate A. A pulsed electric field was applied at 10 kV / cm for 4 min to obtain pulsed electric field pretreated Shatang pomelo homogenate B. The pH of the pulsed electric field pretreated homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.2% (g / g fresh weight of Shatang pomelo raw material) of a pectinase and cellulase (1:1) composite enzyme preparation was added. After stirring in a magnetic water bath at 45 ℃ for 4 h, the enzymes were inactivated at 95 ℃ for 8 min to obtain pulsed electric field pretreated Shatang pomelo homogenate C coupled with pectinase-cellulase enzymatic hydrolysis. After cooling, the pH was adjusted to 4.5 with 1 M sodium citrate solution. 0.3% (g / g fresh weight of Shatang pomelo raw material) of naringinase was added, and enzymatic hydrolysis was continued at 45 ℃ for 4 h. Then, the enzyme was inactivated at 95 ℃ for 8 min to obtain Shatang pomelo homogenate D after pulsed electric field pretreatment coupled with pectinase-cellulase-naringinase enzymatic hydrolysis. Shatang pomelo homogenate D was evenly spread in a container to a thickness of 1.2 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain low-bitterness Shatang pomelo whole fruit powder enriched with flavonoids and SDF.

[0042] Example 3:

[0043] After removing the oil cell layer and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatang pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatang pomelo homogenate A. A pulsed electric field was applied at 8 kV / cm for 5 min to obtain pulsed electric field pretreated Shatang pomelo homogenate B. The pH of the pulsed electric field pretreated homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.1% (g / g fresh weight of Shatang pomelo raw material) of a pectinase and cellulase (1:1) composite enzyme preparation was added. After stirring in a magnetic water bath at 50 ℃ for 2 h, the enzymes were inactivated at 105 ℃ for 5 min to obtain pulsed electric field pretreated Shatang pomelo homogenate C coupled with pectinase-cellulase enzymatic hydrolysis. After cooling, the pH was adjusted to 4.5 with 1 M sodium citrate solution. 0.38% (g / g fresh weight of Shatang pomelo raw material) of naringinase was added, and enzymatic hydrolysis was continued at 55 ℃ for 3 h. Then, the enzyme was inactivated at 105 ℃ for 5 min to obtain Shatang pomelo homogenate D after pulsed electric field pretreatment coupled with pectinase-cellulase-naringinase enzymatic hydrolysis. This Shatang pomelo homogenate D was evenly spread in a container to a thickness of 1 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain low-bitterness Shatang pomelo whole fruit powder enriched with flavonoids and SDF.

[0044] Example 4:

[0045] After removing the oil cell layer and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatang pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatang pomelo homogenate A. A pulsed electric field was applied at 10 kV / cm for 3 min to obtain pulsed electric field pretreated Shatang pomelo homogenate B. The pH of the pulsed electric field pretreated homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.1% (g / g fresh weight of Shatang pomelo raw material) of a pectinase and cellulase (1:1) composite enzyme preparation was added. After stirring in a magnetic water bath at 50 ℃ for 2 h, the enzymes were inactivated at 85 ℃ for 10 min to obtain pulsed electric field pretreated Shatang pomelo homogenate C coupled with pectinase-cellulase enzymatic hydrolysis. After cooling, the pH was adjusted to 4.5 with 1 M sodium citrate solution. 0.38% (g / g fresh weight of Shatin pomelo raw material) of naringinase was added, and enzymatic hydrolysis was continued at 50 ℃ for 3 h. Then, the enzyme was inactivated at 85 ℃ for 10 min to obtain Shatin pomelo homogenate D after pulsed electric field pretreatment coupled with pectinase-cellulase-naringinase enzymatic hydrolysis. This Shatin pomelo homogenate D was evenly spread in a container to a thickness of 1.5 cm, vacuum freeze-dried for 72 h, pulverized, and passed through a 40-mesh sieve to obtain low-bitter Shatin pomelo whole fruit powder enriched with flavonoids and SDF. This is Shatin pomelo whole fruit powder (PEF-PCNE-SWFP) pretreated with pulsed electric field coupled with pectinase-cellulase-naringinase enzymatic hydrolysis.

[0046] Comparative Example 1:

[0047] After removing the oil cells and seeds from the Shatin pomelo, the white peel, mesentery, and pulp tissue are collected to obtain Shatin pomelo raw material. Distilled water is added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatin pomelo homogenate. The Shatin pomelo homogenate is evenly spread in a container to a thickness of 1.5 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain Shatin pomelo whole fruit powder (SWFP).

[0048] Comparative Example 2:

[0049] After removing the oil cells and seeds from the Shatin pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatin pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatin pomelo homogenate. The pH of the pure Shatin pomelo homogenate was adjusted to 4.5 with 1 M citric acid solution, and 0.38% (g / g fresh weight of Shatin pomelo raw material) of naringinase was added. The mixture was stirred in a magnetic water bath at 50 ℃ for 3 h, and then the enzyme was inactivated at 85 ℃ for 10 min to obtain Shatin pomelo homogenate hydrolyzed with naringinase. The Shatin pomelo homogenate was evenly spread in a container to a thickness of 1.5 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain Shatin pomelo whole fruit powder hydrolyzed with naringinase (NE-SWFP).

[0050] Comparative Example 3:

[0051] After removing the oil cells and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected to obtain Shatang pomelo raw material. Distilled water was added at a material-to-liquid ratio of 1:4 (w / v), and homogenized to obtain Shatang pomelo homogenate. A pulsed electric field was applied at 10 kV / cm for 3 min to obtain pulsed electric field pretreated Shatang pomelo homogenate. The pH of the pulsed electric field pretreated homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.38% (g / g fresh weight of Shatang pomelo raw material) of naringinase was added, and the mixture was stirred in a magnetic water bath at 50 ℃ for 3 h. The enzyme was then inactivated at 85 ℃ for 10 min to obtain the pulsed electric field pretreated Shatang pomelo homogenate coupled with naringinase hydrolysis. This Shatang pomelo homogenate was evenly spread in a container to a thickness of 1.5 cm, freeze-dried under vacuum for 72 h, pulverized, and passed through a 40-mesh sieve to obtain pulsed electric field pretreated Shatang pomelo whole fruit powder coupled with naringinase hydrolysis (PEF-NE-SWFP).

[0052] Comparative Example 4:

[0053] After removing the oil cell layer and seeds from the Shatang pomelo, the white peel, pericarp, and pulp tissue were collected and mixed with distilled water at a material-to-liquid ratio of 1:4 (w / v) to obtain a Shatang pomelo homogenate. The pH of the pure Shatang pomelo homogenate was adjusted to 4.5 with 1 M citric acid solution. 0.1% (g / g fresh weight of Shatang pomelo raw material) of a pectinase and cellulase (1:1) compound enzyme preparation was added. The mixture was stirred in a magnetic water bath at 50 ℃ for 2 h, and then the enzymes were inactivated at 85 ℃ for 10 min to obtain a Shatang pomelo homogenate hydrolyzed by pectinase and cellulase. After cooling, the pH was adjusted to 4.5 with 1 M sodium citrate solution, and 0.38% (g / g fresh weight of Shatang pomelo raw material) of naringinase was added. The mixture was stirred in a magnetic water bath at 50 ℃ for 3 h, and then the enzymes were inactivated at 85 ℃ for 10 min to obtain a Shatang pomelo homogenate hydrolyzed by pectinase-cellulase-naringinase. The pomelo homogenate was evenly spread in a container to a thickness of 1.5 cm, vacuum freeze-dried for 72 h, pulverized, and passed through a 40-mesh sieve to obtain pomelo whole fruit powder (PCNE-SWFP) hydrolyzed by pectinase-cellulase-naringinase.

[0054] 10 g of each of the whole fruit powders obtained in Examples 1-4 and Comparative Examples 1-4 were taken, and their bitterness was evaluated. The contents of total flavonoids, SDF, IDF and TDF were analyzed. The results are shown in Table 1.

[0055] Table 1. Bitterness value, debittering rate, total flavonoids, SDF, IDF and TDF content, and IDF / SDF ratio of whole fruit powder obtained by different treatment methods for Shatin pomelo.

[0056] Fruit powder sample bitterness level Debitterness removal rate (%) Total flavonoids (mg CE / g DW) SDF (%) IDF (%) TDF (%) IDF / SDF Example 1 0.64±0.12e 88.0±0.6a 18.5±0.3b 17.0±0.8a 31.6±1.0d 48.6±1.8 1.9±0.1d Example 2 0.68±0.16e 87.3±1.2a 18.0±0.1b 16.6±0.8a 31.4±1.2d 48.0±2.0 1.9±0.1d Example 3 0.73±0.22e 86.4±1.8a 17.4±0.6b 16.2±0.4a 31.6±1.5d 47.8±2.1 2.0±0.1d Example 4 0.62±0.12e 88.4±1.2a 18.0±0.3b 17.0±0.4a 31.6±0.6d 48.6±1.0 1.9±0.1d Comparative Example 1 5.35±0.18a / 23.5±1.0a 7.9±0.4d 39.1±1.0a 47.1±1.4 4.9±0.1a Comparative Example 2 3.95±0.21b 26.2±1.5d 22.5±0.7a 10.3±0.3c 37.2±0.8b 47.5±1.1 3.6±0.1b Comparative Example 3 3.14±0.12c 41.3±0.8c 19.4±0.2b 11.1±0.5c 36.9±0.4b 48.0±0.9 3.3±0.1b Comparative Example 4 1.67±0.09d 68.8±0.9b 18.5±0.3b 14.8±0.6b 33.4±1.0c 48.2±1.6 2.3±0.1c

[0057] The results in Table 1 above indicate that the whole fruit powder of Shatang pomelo prepared by pulsed electric field pretreatment coupled with pectinase-cellulase-naringinase enzymatic hydrolysis has the best debittering effect, and has a higher SDF content and a more balanced IDF / SDF ratio.

[0058] Example 5:

[0059] 1) To investigate the effect of pulsed electric field pretreatment parameters on the release of bitter flavonoid glycosides in pomelo homogenate, different electric field strengths, pulse frequencies, and treatment times were set to treat the pomelo homogenate obtained in step one with pulsed electric field. Subsequently, pectinase and cellulase were used for enzymatic hydrolysis according to the method in step three. The juice yield, soluble pectin content, and total flavonoid release rate of the homogenate after pulsed electric field pretreatment and combined enzymatic hydrolysis of pectinase and cellulase were analyzed. The results are shown in Table 2.

[0060] Table 2. Effects of different pulsed electric field pretreatment coupled with pectinase-cellulase hydrolysis on juice yield, soluble pectin content and total flavonoid release rate of Shatang pomelo homogenate.

[0061] sample Electric field strength (kV / cm) Pulse frequency (Hz) Processing time (min) Juice yield (%) Soluble pectin content (mg Gal / g FW) Total flavonoid release rate (%) Comparison 0 0 0 51.9±3.1c 2.5±0.1c 48.6±1.6c 1 5 100 1 72.5±1.5b 3.8±0.2b 70.3±2.3b 2 8 300 3 80.3±1.7a 5.2±0.2a 80.5±1.8a 3 12 500 5 83.9±0.8a 5.4±0.2a 83.0±2.6a 4 15 700 7 84.8±1.1a 5.5±0.3a 84.3±1.5a

[0062] Note: The control sample is the pomelo homogenate prepared according to step one. Samples 1 to 4 are pomelo homogenates obtained by pretreating the pomelo homogenate prepared in step one with different parameters of pulsed electric field and then hydrolyzing it with pectinase and cellulase according to the method in step three. When the letters next to the numbers in the same column are completely different, it indicates that the difference is statistically significant (p < 0.05), and the same applies below.

[0063] Based on the results in Table 2 above, and taking into account both effectiveness and energy consumption, the preferred pulse electric field processing parameters for this invention are: electric field strength of 8~12 kV / cm, pulse frequency of 300~500 Hz, and processing time of 3~5 min.

[0064] 2) After pretreatment with a pulsed electric field coupled with pectinase-cellulase enzymatic hydrolysis to release a large amount of bitter flavonoid glycosides from the whole fruit homogenate of Shatang pomelo, the effects of naringinase hydrolysis parameters on the conversion of bitter flavonoid glycosides were investigated. Single-factor experiments were conducted to optimize the amount of naringinase added, hydrolysis time, and hydrolysis temperature, initially determining the suitable range for each parameter. Subsequently, the Box-Behnken response surface methodology was used to optimize the above three parameters. The total degradation rate of the three main bitter flavonoids (naringin, melitidin, and cigranoside B) in the homogenate after naringinase hydrolysis was used as the optimization index. The effects of the interactions among the factors on the total degradation rate of the main bitter flavonoids are as follows: Figure 1 As shown.

[0065] Response surface methodology (RSM) optimization results showed that the optimal enzymatic hydrolysis conditions for naringinase were: enzyme dosage of 0.38%, hydrolysis time of 2.99 h, and hydrolysis temperature of 49.13 ℃. Under these conditions, the predicted total degradation rate of the main bitter flavonoids was 41.27%. For ease of practical operation, the parameters were adjusted to enzyme dosage of 0.38%, hydrolysis time of 3.0 h, and hydrolysis temperature of 50 ℃. A verification experiment was conducted under these conditions, and the total degradation rate of the main bitter flavonoids in the homogenate after hydrolysis was measured to be 39.56%, which is close to the predicted value with a relative error of less than 5%, proving the reliability of the RSM optimization results.

[0066] A suitable amount of Shatang pomelo homogenate that has been pretreated with a pulsed electric field and coupled with pectinase-cellulase hydrolysis was taken. Different enzyme addition amounts, hydrolysis times and hydrolysis temperatures were set near the optimal naringinase hydrolysis parameters for naringinase hydrolysis. The total degradation rate of the main bitter flavonoids in the homogenate after hydrolysis was measured. The results are shown in Table 3.

[0067] Table 3. Effects of different naringinase hydrolysis parameters on the total degradation rate of main bitter flavonoids in Shatang pomelo homogenate after pulsed electric field pretreatment coupled with pectinase-cellulase hydrolysis.

[0068] Enzymatic hydrolysis samples Enzyme addition amount (%) Enzymatic hydrolysis time (h) Enzymatic hydrolysis temperature (°C) Total degradation rate (%) of major bitter flavonoids 1 0.3 3 45 37.1±0.5 2 0.35 3.5 50 39.4±0.3 3 0.4 4 55 38.8±0.2

[0069] Based on the results in Table 3 above, the preferred enzymatic hydrolysis parameters for this invention are: enzyme addition amount 0.3~0.4%, hydrolysis time 3~4 h, and hydrolysis temperature 45~55 ℃.

[0070] Example 6:

[0071] This embodiment uses an in vitro fecal microbiota fermentation model to anaerobic ferment SWFP (skin-free fermented pomelo powder) prepared in Comparative Example 1, PCNE-SWFP (skin-free fermented pomelo powder) prepared in Comparative Example 4, and PEF-PCNE-SWFP (skin-free fermented pomelo powder with low bitterness and retained active ingredients) prepared in Example 4 for 24 h. Gas chromatography was used to analyze the content of beneficial metabolites (SCFAs) such as acetic acid, propionic acid, and butyric acid in the fermentation broth of each group. It was found that SWFP, PCNE-SWFP, and PEF-PCNE-SWFP all significantly increased the yield of the aforementioned SCFAs, with PEF-PCNE-SWFP showing a more significant effect, particularly in promoting butyric acid production, with its butyric acid content being 2.2 and 1.6 times higher than that of the former, respectively (e.g., ...). Figure 2 (As shown). The content of lipopolysaccharide (LPS), a harmful metabolite of microbial flora, in fermentation broth was detected using a lipopolysaccharide enzyme-linked immunosorbent assay (ELISA) kit. It was found that PEF-PCNE-SWFP was more effective than SWFP and PCNE-SWFP in reducing LPS levels, with LPS levels being 0.6 and 0.7 times higher, respectively (e.g., as shown). Figure 3 (As shown).

[0072] Therefore, the whole fruit powder of Shatin pomelo (PEF-PCNE-SWFP) prepared by pulsed electric field coupling of pectinase-cellulase-naringinase is more effective than the original fruit powder (SWFP) and the whole fruit powder of Shatin pomelo (PCNE-SWFP) prepared by pectinase-cellulase-naringinase enzymatic hydrolysis in promoting the production of beneficial metabolites SCFAs and inhibiting the production of harmful metabolites LPS by intestinal flora. It shows a better intestinal microecological regulation effect and can be used as a health food ingredient to improve intestinal microecological disorder and for the development of related health foods.

[0073] Those skilled in the art can make appropriate adjustments within the scope of the principles and core parameters of this invention, and these adjustments should also be considered to fall within the protection scope of this invention.

Claims

1. A method for preparing a low-bitterness Shatin pomelo whole fruit powder that improves intestinal microecological disorder, characterized in that, Includes the following steps: Step 1: Peel off the oil cells from the pomelo, cut it into small pieces and remove the seeds to obtain the raw pomelo material; Add distilled water to it and beat it to obtain a smooth and free-flowing pomelo homogenate A; Step 2: Take the pomelos homogenate A obtained in Step 1 and treat it with a pulsed electric field to obtain pomelos homogenate B pretreated with a pulsed electric field. Step 3: Take the pomelo homogenate B obtained in Step 2, add pectinase and cellulase, and stir the reaction in a water bath; after the reaction is complete, inactivate the enzymes to obtain pomelo homogenate C after enzymatic hydrolysis by pectinase and cellulase. Step 4: Take the pomelo homogenate C obtained in Step 3, add naringinase, and stir the reaction in a water bath; after the reaction is complete, inactivate the enzyme to obtain pomelo homogenate D after naringinase hydrolysis. Step 5: Vacuum freeze-dry the pomelo homogenate D obtained in Step 4, then pulverize and sieve it to obtain pomelo whole fruit powder.

2. The preparation method according to claim 1, characterized in that, in In step two, the pomelo homogenate A is subjected to pulsed electric field treatment for 3 to 5 minutes under the conditions of electric field strength of 8~12 kV / cm and pulse frequency of 300~500 Hz to obtain pomelo homogenate B pretreated by pulsed electric field.

3. The preparation method according to claim 2, characterized in that, In step three, the pH of the Shatin pomelo homogenate B is adjusted to 4.0-5.0 with citric acid solution, and 0.1-0.2% of a compound enzyme preparation of pectinase and cellulase is added. The mixture is stirred and reacted in a water bath at 45-55 ℃ for 2-4 h. Then the enzymes are inactivated to obtain Shatin pomelo homogenate C after enzymatic hydrolysis by pectinase and cellulase. The weight ratio of pectinase to cellulase is 1:

1.

4. The preparation method according to claim 3, characterized in that, In step four, the pH of the Shatin pomelo homogenate C was adjusted to 4.0-5.0 with sodium citrate solution, 0.3-0.4% naringinase was added, and the mixture was stirred in a water bath at 45-55 ℃ for 3-4 h. Then the enzyme was inactivated to obtain Shatin pomelo homogenate D after naringinase hydrolysis.

5. The preparation method according to claim 4, characterized in that, In step one, distilled water is added to the Shatin pomelo raw material at a material-to-liquid ratio of 1:3 to 1:5 to make a pulp, resulting in a Shatin pomelo homogenate A with good fluidity.

6. The preparation method according to claim 5, characterized in that, in In step five, the Shatin pomelo homogenate D is evenly spread in a container to a thickness of 1-1.5 cm, and then placed in a freeze dryer for vacuum freeze drying until the moisture content is below 5%. After that, it is pulverized and passed through a 40-mesh sieve to obtain Shatin pomelo whole fruit powder.

7. A low-bitterness pomelos whole fruit powder for improving intestinal microecological disorder, prepared by the method described in claim 1.

8. The use of the whole fruit powder of pomelo as described in claim 7 in the preparation of food or health products that improve intestinal microecological disorders.

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