A raw and ripe polygonatum multiflorum mixed fermentation liquid product and a preparation method thereof

Abstract

The application discloses a kind of raw and cooked rhizoma polygonati complex fermentation liquor products and preparation method thereof, belong to the field of biotechnology.The application provides a kind of method for fermenting complex rhizoma polygonati, the method includes the following steps: (1) raw rhizoma polygonati powder and cooked rhizoma polygonati powder are mixed after sterilization;(2) after adding microbial inoculum to the mixture after sterilization, fermentation is carried out, to obtain complex rhizoma polygonati;The microbial inoculum is lactobacillus casei liquid, lactobacillus plantarum liquid, lactococcus lactis liquid or yeast bacteria liquid;The application provides a kind of method for conveniently preparing rhizoma polygonati fermentation liquor, the color of the obtained fermentation liquor is clear and bright, aroma is added and rich, sweet and mellow in taste, greatly improve the quality of rhizoma polygonati fermentation liquor, and a variety of nutrients in the complex rhizoma polygonati fermentation liquor after fermentation are significantly improved.

Description

Technical Field

[0001] This invention relates to a mixed fermentation liquid product of raw and cooked Polygonatum sibiricum and its preparation method, belonging to the field of biotechnology. Background Technology

[0002] Polygonatum sibiricum is a perennial herb belonging to the genus Polygonatum in the family Liliaceae. Its thick, fleshy, nodular rhizomes are the main part used. The rhizomes are rich in polysaccharides, saponins, flavonoids, and alkaloids, possessing the effects of tonifying qi and nourishing yin, strengthening the spleen and moistening the lungs, and tonifying the kidneys and replenishing essence. Simultaneously, its rich content of starch, fat, protein, carotene, and vitamins makes it suitable as a food source. As a dual-purpose medicinal and edible resource in my country, Polygonatum has been proven to have antioxidant, anti-inflammatory, blood sugar and lipid-regulating, immune-enhancing, and tumor-suppressing effects. However, raw Polygonatum can irritate the throat and skin, causing numbness in the mouth and tongue when consumed. While steaming enhances its health benefits, it also dramatically increases its sour, bitter, and astringent taste, resulting in a strong medicinal flavor and a sticky texture, negatively impacting the development of Polygonatum products and leading to poor consumer acceptance. Improving the flavor, sensory qualities, and nutritional properties of Polygonatum, enhancing its bioavailability, and expanding its applications in food are urgent problems that need to be addressed.

[0003] Polygonatum rhizome, rich in polysaccharides, offers natural advantages for microbial fermentation. Microbial fermentation of natural plant resources can alter the composition of raw materials, thus affecting their flavor. Furthermore, under the action of enzymes produced by microbial metabolism, it can break down cell walls, promote the dissolution of active substances, or achieve structural modification and transformation of certain active components in the substrate, thereby improving its bioavailability. Simultaneously, raw and cooked Polygonatum rhizome have distinct flavors and significant differences in composition. Compound fermentation of Polygonatum rhizome can fully utilize its own components to balance flavor, neutralize taste, and balance nutritional components without introducing additional additives. Moreover, the fermentation process is conducted under mild conditions, facilitating large-scale production without significantly increasing costs. The resulting fermented broth has significant health benefits.

[0004] Existing technologies have included research on fermented products made from Polygonatum sibiricum. For example, CN106983054A discloses a Polygonatum sibiricum lactic acid fermented health beverage and its preparation method, which includes: extracting pretreated Polygonatum sibiricum, adding sugars to the obtained Polygonatum sibiricum extract or extract filtrate, fermenting the mixture with lactic acid bacteria, and filtering, blending, sterilizing, and cooling the obtained fermented filtrate to obtain the final product. Although this method emphasizes that using lactic acid bacteria fermentation helps preserve the nutrients in Polygonatum sibiricum, fully utilizes its nutritional effects, and gives the beverage the characteristics of a lactic acid bacteria beverage, inhibiting the growth of harmful microorganisms and preventing diarrhea, this beverage contains high levels of lactose, fructose, sucrose, and other sugars, as well as exogenous substances such as citric acid and β-cyclodextrin. While positioned as a health beverage, it is not suitable for diabetic patients.

[0005] CN105331487A discloses a method for preparing a fermented health beverage made from Polygonatum sibiricum, including: pretreatment, extraction, fermentation, clarification, flavoring, sterilization, and post-ripening of fresh Polygonatum sibiricum. This alcoholic fermented health beverage is prepared by alcoholic fermentation, and its flavor is significantly different from beverages obtained through lactic acid fermentation. Although it emphasizes that this fermentation method facilitates the rapid and full absorption of beneficial components in Polygonatum sibiricum by the human body, achieving the goals of tonifying qi and kidneys, delaying aging, and preventing sub-health, this beverage is still an alcoholic fermented beverage and contains a certain amount of alcohol, thus limiting its consumer base. Meanwhile, there are also fermented products made from Polygonatum sibiricum combined with other ingredients, such as CN104223271B, which discloses a fermented beverage made from okra with lactic acid bacteria; CN105918751A, which discloses a fermented beverage made from Cordyceps sinensis with lactic acid bacteria; and CN1738905A, which discloses a yogurt product containing fermented ginseng with lactic acid bacteria.

[0006] However, existing processes involve cumbersome raw material handling, and the resulting byproducts are unfriendly to certain populations. This invention features simple raw material handling requirements, mild processing conditions, low environmental and equipment requirements, no external additives, naturally suitable product quality for a wide range of consumers, and the potential for large-scale production. Summary of the Invention

[0007] Polygonatum sibiricum has both medicinal and edible uses. Fermentation with probiotics can, to some extent, improve the absorption of its beneficial components, enhance the flavor and nutritional content of Polygonatum sibiricum products, and thus better meet people's demand for natural health benefits. However, different fermentation agents have different metabolic capacities and pathways, and the overall flavor quality and beneficial component composition of fermented products mainly depend on the type of fermentation agent. Therefore, screening, optimizing, and identifying the most suitable flavor-enhancing fermentation bacteria and fermentation processes for Polygonatum sibiricum is of significant academic and economic importance for developing fermented Polygonatum sibiricum foods and beverages.

[0008] To address the issues of existing fermented Polygonatum products having a strong medicinal aroma and bitter taste, requiring excessive external flavoring substances, and being unsuitable for certain populations, this application explores the use of different probiotics to ferment Polygonatum. This probiotic fermentation alters the internal components of Polygonatum, thereby improving its flavor, quality, and health benefits. Optimal probiotic fermentation can reduce unpleasant flavors, and the combination of raw and cooked Polygonatum can better neutralize unpleasant sensory characteristics while preserving beneficial components to the greatest extent. Probiotic fermentation hydrolyzes large-molecule polysaccharides, enhancing flavor while promoting the formation of easily absorbed small-molecule prebiotics.

[0009] This invention provides a method for fermenting compound Polygonatum, the method comprising the following steps:

[0010] (1) Sterilize and cool the raw and cooked Polygonatum sibiricum mixture;

[0011] (2) Fermentation is carried out after adding microbial agents to the sterilized mixture; the microbial agents are Lactobacillus casei liquid, Lactobacillus plantarum liquid, Streptococcus thermophilus liquid, Lactococcus lactis liquid, kombucha liquid or yeast liquid.

[0012] In one embodiment of the present invention, the concentration of the microbial agent is at least 1 × 10⁻⁶. 7 CFU / mL.

[0013] In one embodiment of the present invention, step (1) involves mixing raw and cooked Polygonatum sibiricum in a mass ratio of 4:1 to 1:4.

[0014] In one embodiment of the present invention, step (1) is to mix raw Polygonatum and cooked Polygonatum in a mass ratio of 1:1.

[0015] In one embodiment of the present invention, the mixture of raw and cooked Polygonatum is added to the fermentation system at a mass ratio of 2% to 4%, i.e., 2g / 100mL to 4g / 100mL.

[0016] In one embodiment of the present invention, in step (2), the amount of microbial agent added is 1-4% (v / v).

[0017] In one embodiment of the present invention, in step (2), the fermentation conditions are as follows: after adding microbial agents to the sterilized mixture, the fermentation system is sterilized by ultraviolet light for 20-30 minutes, fermented at 35-40°C for 6-8 hours, and then aged at 2-4°C for 10-12 hours.

[0018] In one embodiment of the present invention, the sterilization method in step (1) includes, but is not limited to, high-pressure steam sterilization.

[0019] In one embodiment of the present invention, the preparation method of the Lactobacillus casei culture, Lactobacillus plantarum culture, Streptococcus thermophilus culture, Lactococcus lactis culture, kombucha culture, and yeast culture is as follows:

[0020] (1) Lactobacillus casei, Lactobacillus plantarum, Streptococcus thermophilus, Lactococcus lactis, kombucha, and yeast were inoculated into M17 medium and cultured at 35-37℃ respectively; Lactobacillus casei seed liquid, Lactobacillus plantarum seed liquid, Streptococcus thermophilus seed liquid, Lactococcus lactis seed liquid, kombucha seed liquid, and yeast seed liquid were prepared respectively.

[0021] (2) The prepared Lactobacillus casei seed solution, Lactobacillus plantarum seed solution, Streptococcus thermophilus seed solution, Lactococcus lactis seed solution, kombucha seed solution, and yeast seed solution were inoculated into MRS broth medium at an inoculation rate of 1-10% (v / v) and cultured at 35-37℃ and 150-200 r / min for 12-16 h to obtain the culture solutions respectively.

[0022] (3) The obtained culture solutions were centrifuged at 3000-5000 r / min and 2-4℃ for 5-10 min to collect the bacterial cells. Then, after dilution with physiological saline, Lactobacillus casei culture solution, Lactobacillus plantarum culture solution, Streptococcus thermophilus culture solution, Lactococcus lactis culture solution, Kombucha culture solution, and yeast culture solution were obtained respectively.

[0023] In one embodiment of the present invention, step (2) involves inoculating the prepared Lactobacillus casei seed solution, Lactobacillus plantarum seed solution, Streptococcus thermophilus seed solution, Lactococcus lactis seed solution, kombucha seed solution, and yeast seed solution into MRS broth at an inoculation rate of 1-10% (v / v) and pre-culturing them at 37°C and 200 r / min for 16 h to obtain culture solutions.

[0024] In one embodiment of the present invention, step (3) involves centrifuging the culture medium obtained in step (2) at 5000 r / min and 4℃ for 5 min to collect the bacterial cells, and then diluting it with physiological saline to obtain a viable bacterial count of approximately 10. 7 CFU / mL single-strain bacterial culture.

[0025] In one embodiment of the present invention, the raw Polygonatum is: selected fresh Polygonatum that has been pre-dried, washed and de-haired, sliced ​​with a thickness of 2-5 mm, and naturally air-dried to obtain raw Polygonatum slices with a moisture content of ≤6%.

[0026] The prepared Polygonatum is prepared by selecting fresh Polygonatum that has been sun-dried in advance, steaming it in a steamer for 6 hours, turning off the heat and letting it sit for 8 hours, taking it out and placing it in a 60℃ oven until the outer skin is slightly dry. This is the prepared Polygonatum prepared by steaming and sun-drying once. The above method is repeated 9 times for steaming and sun-drying, and then it is cut and dried to obtain prepared Polygonatum prepared by nine steaming and sun-drying with a moisture content of ≤15%.

[0027] The present invention also provides a compound Polygonatum preparation using the above method.

[0028] This invention provides products containing the above-mentioned compound Polygonatum, including food, pharmaceuticals, and health products.

[0029] In one embodiment of the present invention, the food includes a fermented beverage made from Polygonatum sibiricum.

[0030] Beneficial effects

[0031] This invention provides a convenient method for preparing Polygonatum sibiricum fermentation broth. The resulting fermentation broth is clear and bright in color, with a rich and fragrant aroma and a sweet and sour taste, significantly improving the quality of the Polygonatum sibiricum fermentation broth. The fermented compound Polygonatum sibiricum fermentation broth shows a significant increase in various nutrients, with the content of polysaccharides that are difficult to absorb decreasing by 35% compared to the control group. However, the content of easily absorbed monosaccharide components—glucose, galactose, galacturonic acid, and arabinose—increased by 1.3, 1.2, 2.0, and 1.6 times, respectively. The sensory irritation caused by saponins decreased by 76%, and the total amount of prebiotic short-chain fatty acids increased by 1.6 times. Furthermore, the prepared fermentation broth contains no added external aids and no alcohol or other components, making its quality more readily accepted by consumers and beneficial for expanding the consumer base of Polygonatum sibiricum. Attached Figure Description

[0032] Figure 1 The graph shows the changes in polysaccharide content in the fermentation broth of Polygonatum odoratum.

[0033] Figure 2 This is a graph showing the changes in the monosaccharide composition of the compound Polygonatum fermentation broth.

[0034] Figure 3 The graph shows the changes in saponin content in the fermentation broth of Polygonatum odoratum.

[0035] Figure 4 This graph shows the changes in the content of prebiotic products—short-chain fatty acids—in the fermented broth of Polygonatum odoratum fermented by probiotics. Detailed Implementation

[0036] The food fermentation agents used in the following examples were purchased from Beijing Chuanxiu Technology Co., Ltd., and all the monomeric strains used were isolated from this commercial fermentation agent.

[0037] The *Lactobacillus casei*, *Lactobacillus plantarum*, *Streptococcus thermophilus*, *Lactococcus lactis*, kombucha, and *Saccharomyces cerevisiae* strains involved in the following examples are all disclosed in the paper *Enhanced biotransformation of bioactive components and volatile compounds of bamboo (Phyllostachys glauca McClure) leaf juice fermented by probiotic *Streptococcus thermophiles*, LWT, Volume 173, 2023, 114363. For ease of explanation, the above strains are respectively named as: *Lactobacillus casei* 4N, *Lactobacillus plantarum* NH, *Streptococcus thermophilus* IMAU, *Lactococcus lactis* Un, kombucha Tf, and yeast.

[0038] The applicant promises to release the above-mentioned biological materials to the public within twenty years from the date of application: Lactobacillus casei 4N, Lactobacillus plantarum NH, Streptococcus thermophilus IMAU, Lactococcus lactis Un, Kombucha Tf, and yeast.

[0039] The Polygonatum used in the following examples was purchased from Yipuyuan Polygonatum Technology Co., Ltd., Xinhua County, Hunan Province.

[0040] The culture media involved in the following examples are as follows:

[0041] M17 liquid culture medium (product code: AC12076, g / L): soybean peptone 5.0, peptone 2.5, casein peptone 2.5, yeast extract 2.5, beef extract 5.0, lactose 5.0, sodium ascorbate 0.5, sodium β-glycerophosphate 19.0, magnesium sulfate 0.25.

[0042] MRS medium (product code: BNCC370433, g / L): casein peptone 10.0, diammonium citrate 2.0, yeast extract 4.0, beef extract 8.0, glucose 20.0, Tween-80 1.0, magnesium sulfate 0.2, manganese sulfate 0.04, sodium acetate 5.0, dipotassium hydrogen phosphate 2.0.

[0043] The detection methods involved in the following embodiments are as follows:

[0044] Flavor sensory evaluation test:

[0045] Twenty volunteers were selected from among the flavor and sensory volunteers to participate in a flavor and sensory preference test, which comprehensively evaluated and compared the appearance clarity, aroma, and taste of the fermentation broth. The scoring criteria are as follows: 1-5 points.

[0046] Table 1: Sensory Evaluation Criteria

[0047]

[0048] Detection of monosaccharide content:

[0049] Monosaccharide content: Accurately measure 200 μL of fermentation broth into an ampoule, add 2 mL of 3M TFA, and hydrolyze at 120 °C for 3 h. Accurately pipette the acid hydrolysis solution into a tube and blow it dry under nitrogen. Add 1 mL of water and vortex to mix. Pipe 50 μL of the solution into 950 μL of deionized water and centrifuge at 12000 rpm for 5 min. Take the supernatant for IC analysis. Chromatographic column: Dionex Carbopac™ PA20 (3 × 150 mm); mobile phase: A: H2O; B: 15 mM NaOH; C: 15 mM NaOH and 100 mM sodium acetate (NaOAC); flow rate: 0.3 mL / min; injection volume: 5 μL; column temperature: 30 °C; detector: electrochemical detector.

[0050] Saponin measurement:

[0051] According to the vanillin-perchloric acid colorimetric method: 5 mL of Polygonatum sibiricum fermentation broth was added to 80% ethanol and refluxed to a final volume of 50 mL at 70℃ for 2 h. The extraction was repeated twice. The supernatant was collected by centrifugation at 8000 r / min, concentrated by rotary evaporation, and extracted three times with n-butanol at a 1:1 ratio. The extract was then concentrated under reduced pressure to a solid volume to obtain the crude extract of Polygonatum sibiricum saponins. 0.2 g of the crude saponin extract was diluted to a final volume of 100 mL with methanol. A standard curve was prepared using ginsenoside Rb1, and the saponin content in the sample was calculated.

[0052] Short-chain fatty acid content:

[0053] Take an appropriate amount of fermentation broth into a 2 mL centrifuge tube, add 50 μL of 15% phosphoric acid, then add 10 μL of 75 μg / mL internal standard (isohexanoic acid) solution and 140 μL of diethyl ether, homogenize for 1 min, centrifuge at 12000 rpm for 10 min at 4℃, and take the supernatant for analysis. A Thermo Trace 1300 (Thermo Fisher Scientific, USA) gas chromatography system was used, with an Agilent HP-INNOWAX capillary column (30 m × 0.25 mm ID × 0.25 μm); split injection was used, with an injection volume of 1 μL and a split ratio of 10:1. The injection port temperature was 250℃; the ion source temperature was 300℃; and the transfer line temperature was 250℃. The temperature program started at 90℃; then increased to 120℃ at 10℃ / min; then increased to 150℃ at 5℃ / min; and finally increased to 250℃ at 25℃ / min and held for 2 min. Helium was used as the carrier gas at a flow rate of 1.0 mL / min. Mass spectrometry was performed using a Thermo ISQ 7000 mass spectrometer (Thermo Fisher Scientific, USA), with an electron impact ionization (EI) source, SIM scan mode, and an electron energy of 70 eV.

[0054] Polysaccharide content:

[0055] Take 10 mL of the supernatant from the fermentation broth after high-speed centrifugation, add 30 mL of ethanol to precipitate, place in a refrigerator at 4℃ for 24 h, centrifuge at 8000 r / min for 10 min, remove the solution and retain the precipitate; add 10 mL of water to fully dissolve the precipitate, add another 30 mL of ethanol to precipitate, centrifuge for 10 min, remove the supernatant, and add 5 mL of water to dissolve the precipitate to obtain the crude polysaccharide extract. The polysaccharide content extracted from the fermentation broth was determined using the total sugar determination method of phenol sulfate.

[0056] Example 1: Screening of Fermentation Agents

[0057] (1) Preparation of fermentation inoculum (preparation of microbial inoculum)

[0058] The mixed fermentation agent was isolated and precultured in M17 medium supplemented with 1% glucose.

[0059] Lactobacillus casei 4N, Lactobacillus plantarum NH, Streptococcus thermophilus IMAU, Lactococcus lactis Un, Kombucha Tf, and yeast were inoculated into MRS broth at an inoculation rate of 1–10% (v / v) and pre-cultured at 37°C and 200 r / min for 16 h as seed culture.

[0060] The pre-cultured seed culture was centrifuged at 5000 rpm and 4°C for 5 min to collect bacterial cells. After dilution with physiological saline, the viable cell count was approximately 10. 7CFU / mL single-strain bacterial suspension; that is, bacterial concentrations prepared separately at 1×10⁻⁶. 7 The concentration of Lactobacillus casei 4N bacterial suspension (CFU / mL) was 1×10⁻⁶. 7 The concentration of *Lactobacillus plantarum* NH bacterial suspension (CFU / mL) was 1×10⁻⁶. 7 The concentration of Streptococcus thermophilus IMAU bacterial suspension (CFU / mL) was 1×10⁻⁶. 7 The concentration of Lactococcus lactis Un bacterial suspension at CFU / mL was 1×10⁻⁶. 7 The concentration of kombucha Tf culture (CFU / mL) was 1×10⁻⁶. 7 Yeast culture solution with CFU / mL.

[0061] (2) Pretreatment of raw and processed Polygonatum:

[0062] Selected fresh Polygonatum rhizome with good appearance and high quality, which has been pre-dried, is washed and de-haired, then sliced ​​to a thickness of 2-5mm, and naturally air-dried to obtain raw Polygonatum rhizome slices with a moisture content of ≤6%.

[0063] Take another fresh Solomon's Seal, steam it in a steamer for 6 hours, turn off the heat and let it sit for 8 hours, take it out and place it in a 60℃ oven until the outer skin is slightly dry. This is the processed Solomon's Seal after one steaming and drying. Repeat the steaming and drying process 9 times, cut and dry to obtain the processed Solomon's Seal after nine steaming and nine drying, with a moisture content of ≤15%.

[0064] The prepared raw and cooked Polygonatum sibiricum were ground into powder and passed through a 100-mesh sieve for later use.

[0065] (3) Fermented compound Polygonatum

[0066] Raw and cooked Polygonatum were mixed in a mass ratio of 1:1 to obtain 2g of mixed powder. 100mL of deionized water was added to the mixed powder, and the mixture was sealed and placed in a high-pressure steam sterilizer at 121℃ for 15min.

[0067] When the temperature of the Polygonatum sibiricum solution obtained after high-pressure sterilization was lowered to 30℃, 1×10⁻⁶ bacteria were added to each solution. 7 Fermentation was carried out using a bacterial culture of CFU / mL at an inoculum size of 1% (v / v). The fermentation conditions were as follows: the fermentation system was sterilized by UV light for 30 min, fermented at 38℃ for 6 h, and then aged at 4℃ for 12 h. Fermentation broths were obtained separately. Based on sensory evaluation of the fermentation broths, a superior probiotic fermentation agent of compound Polygonatum leaves was selected.

[0068] The sensory evaluation of the Polygonatum sibiricum fermented liquid after fermentation with probiotic starter is shown in Table 2; where A is the mixed fermentation liquid (the above-prepared Lactobacillus casei 4N liquid, Lactobacillus plantarum NH liquid, Streptococcus thermophilus IMAU liquid, Lactococcus lactis Un liquid, Kombucha Tf liquid, and yeast liquid were mixed in a volume ratio of 1:1:1:1:1:1 to obtain a bacterial concentration of 1×10⁻⁶. 7 A) Mixed fermentation broth (CFU / mL); B) Lactobacillus casei fermentation group; C) Lactobacillus plantarum fermentation group; D) Streptococcus thermophilus fermentation group; E) Lactococcus lactis fermentation group; F) Kombucha fermentation group; G) Yeast fermentation group.

[0069] Table 2: Sensory evaluation of compound Polygonatum sibiricum fermentation broth with different probiotic fermentation agents

[0070]

[0071] The results showed that the control group of compound Polygonatum sibiricum liquid without added probiotic fermentation agent was a dark brown suspension mainly composed of cooked Polygonatum sibiricum, with a more obvious medicinal aroma, a distinct bitter taste, and the pungent taste of raw Polygonatum sibiricum.

[0072] The fermented liquid of Polygonatum odoratum fermented with mixed microbial agents is turbid, with white spots of microbial cells on the surface, a strong sour and putrid smell, no medicinal aroma, and a sour and bitter taste.

[0073] The fermentation broth of Polygonatum sibiricum fermented with Lactobacillus casei has improved clarity, has a strong medicinal aroma, but the fragrance is slightly reduced, and it is mainly bitter.

[0074] The fermentation broth of Lactobacillus plantarum is clear, with a strong fermentation flavor and a sweet roasted aroma, and a weak bitter taste, making it the most acceptable.

[0075] The fermentation broth of Streptococcus thermophilus slightly improves clarity, has a milder fermentation flavor, with a sweet aroma and a stronger sour taste;

[0076] Lactococcus lactis fermentation broth is used to improve clarity, but it has the worst acceptability, an off-odor, and a sour and bitter taste.

[0077] Kombucha fermentation liquid has slightly improved clarity, but the fermentation flavor is milder, with a sour and astringent taste and a slightly pungent sensation.

[0078] The yeast fermentation liquid is clear, with a mild fermentation flavor, a sweet aroma, a sweet aroma reminiscent of roasted Solomon's Seal, a strong sour taste, and a lingering bitterness.

[0079] Therefore, based on comprehensive sensory evaluation, *Lactobacillus plantarum* was selected as the microbial fermentation agent for compound *Polygonatum sibiricum* fermentation products.

[0080] Example 2: Sensory effects of fermented raw and cooked Polygonatum sibiricum by preferred probiotics

[0081] The raw and cooked Polygonatum were fermented separately using Lactobacillus plantarum. The specific implementation method was the same as in Example 1, except that step (3) was adjusted as follows:

[0082] Replace 2g of the mixed powder of raw and cooked Polygonatum in a 1:1 mass ratio with an equal mass of raw or cooked Polygonatum powder, and repeat the following steps: add 100mL of deionized water to either raw or cooked Polygonatum powder, seal and sterilize in a 121℃ autoclave for 15min; when the temperature of the Polygonatum solution obtained after autoclaving drops to 30℃, add 1×10⁻⁶ bacteria obtained in step (1) to each solution. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture at an inoculum size of 1% (v / v). The fermentation conditions were as follows: the above fermentation system was sterilized by ultraviolet light for 30 min, fermented at 38℃ for 6 h, and then aged at 4℃ for 12 h to obtain fermentation broth.

[0083] Meanwhile, unfermented raw Polygonatum and unfermented cooked Polygonatum were used as controls; the sensory evaluation of the fermentation broth of raw / cooked Polygonatum after fermentation with Lactobacillus plantarum was shown in Table 3 to evaluate the effect of fermentation of raw / cooked Polygonatum alone on flavor quality.

[0084] Table 3: Sensory evaluation of fermented broth containing Lactobacillus plantarum and Polygonatum sibiricum

[0085]

[0086] The results showed that adding Lactobacillus plantarum for fermentation can improve the clarity of Polygonatum sibiricum.

[0087] Raw Polygonatum sibiricum undergoes fermentation, resulting in a strong sour and rotten taste, severe foul odor, and a strong numbing and irritating sensation on the tongue.

[0088] Cooked Polygonatum has a natural sweet and medicinal aroma. After fermentation, the aroma is improved, with a slight fermented aroma and medicinal smell. The taste is also greatly improved, being more sour and the bitterness is significantly reduced.

[0089] Due to the significant differences in the composition of raw and cooked Polygonatum, fermentation with Lactobacillus plantarum has different effects on its flavor and quality. Fermentation of cooked Polygonatum alone is more effective in improving the flavor than that of raw Polygonatum, but the overall flavor improvement is less significant compared to that of mixed Polygonatum.

[0090] Example 3: Optimization of liquid fermentation conditions for compound Polygonatum powder

[0091] Under the condition of screening suitable fermentation bacteria, the effects of fermentation conditions such as the ratio and amount of raw and cooked Polygonatum sibiricum, the amount of fermentation bacteria added, fermentation temperature, and fermentation time on the flavor and quality of Polygonatum sibiricum fermentation liquid were further investigated.

[0092] The specific steps are as follows:

[0093] 1. The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0094] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they were mixed in proportions of 4:1, 2:1, 1:1, 1:2, and 1:4 (the total amount added was 2g). 100mL of deionized water was added to the mixed Polygonatum sibiricum powder, and the mixture was sealed and placed in a high-pressure steam sterilizer at 121℃ for 15min. Meanwhile, pure raw Polygonatum sibiricum powder and pure cooked Polygonatum sibiricum powder were used as controls.

[0095] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, 2 mL (2%) of the bacterial concentration obtained in step (1) was added respectively, and the concentration was 1×10. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 35℃ for 6 h, and then aged at 4℃ for 12 h to obtain fermentation liquid. The changes in clarity, aroma and taste of the fermentation liquid were evaluated.

[0096] The sensory evaluation of the optimized fermentation broth treated with a mixture of raw and cooked Polygonatum sibiricum powder is shown in Table 4.

[0097] Table 4: Sensory evaluation of different raw and cooked Polygonatum sibiricum powder fermentation broths

[0098]

[0099]

[0100] The results showed that pure raw Polygonatum sibiricum fermentation had a strong stimulating sensation, numbness on the tongue, a strong raw starchy smell, a fermented sour odor, and a bland taste; while pure mature Polygonatum sibiricum powder fermentation had a strong medicinal smell, a severe bitter taste, and a weak fermented flavor.

[0101] Compared to adding different blending ratios, when adding raw Polygonatum to cooked Polygonatum in a ratio of 1:2, the fermentation liquid is clearer, the fermentation aroma is richer, the taste is moderately sweet and sour, and the bitterness is significantly reduced.

[0102] 2. The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0103] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they were mixed at a ratio of raw Polygonatum sibiricum powder to cooked Polygonatum sibiricum powder of 1:2. The mixture was added to 100mL of deionized water at material-to-liquid ratios of 1:100, 1:50 and 1:25 (the addition amounts were 1g, 2g and 4g respectively). The mixture was then sealed and placed in a high-pressure steam sterilizer at 121℃ for 15min for sterilization.

[0104] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, 2 mL of the bacterial concentration obtained in step (1) was added to each solution. The bacterial concentration of each solution was 1×10⁻⁶. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 35℃ for 6 h, and then aged at 4℃ for 12 h to obtain fermentation liquid. The changes in clarity, aroma and taste of the fermentation liquid were evaluated.

[0105] The sensory evaluation of the effect of the amount of raw and cooked Polygonatum sibiricum compound powder on the fermentation juice is shown in Table 5.

[0106] Table 5: Sensory evaluation of the amount of compound powder added from Polygonatum sibiricum fermentation broth

[0107]

[0108] The results showed that the fermentation liquid with an additive-to-liquid ratio of 1:50 was clear and uniform, with a rich fermentation flavor, sweet aroma, and weak bitterness; the fermentation liquid with an additive-to-liquid ratio of 1:100 had a bland taste, weak fermentation aroma, and strong starchy smell compared to the 1:50 ratio; the fermentation liquid with an additive-to-liquid ratio of 1:25 was turbid, had a strong medicinal smell, a heavy bitterness, and poor flavor acceptability.

[0109] 3. The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0110] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they were mixed in a ratio of raw Polygonatum sibiricum powder to cooked Polygonatum sibiricum powder of 1:2. 100 mL of deionized water was added to the mixed Polygonatum sibiricum powder, and the mixture was sealed and placed in a high-pressure steam sterilizer at 121℃ for 15 min for sterilization. The ratio of raw to cooked Polygonatum sibiricum added was 1:50, and the total amount was 2 g.

[0111] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, the bacterial concentrations obtained in step (1) were inoculated at volume ratios of 1% (1mL), 2% (2mL), and 4% (4mL), respectively. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 35℃ for 6 h, and then aged at 4℃ for 12 h to obtain fermentation broth. The clarity, aroma and taste changes of the fermentation broth were evaluated.

[0112] The effects of the amount of *Lactobacillus plantarum* added are shown in Table 6.

[0113] Table 6: Sensory evaluation of Lactobacillus plantarum starter culture inoculum amount

[0114]

[0115] The results showed that when the amount of Lactobacillus plantarum added was 1%, the fermentation characteristics were not obvious, the medicinal smell was strong, the bitter taste was obvious, and the irritation was strong. When the amount of Lactobacillus plantarum added was 2%, the fermentation flavor was significant, sweet and fragrant, with a moderate sweet and sour taste, no bitter taste, and good acceptability. When the amount of Lactobacillus plantarum added was 4%, the fermentation liquid was turbid, had a sour smell, was severely sour, had a strong astringent taste, and the fermentation flavor was poorly accepted.

[0116] 4. The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0117] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they were mixed in a ratio of raw Polygonatum sibiricum powder to cooked Polygonatum sibiricum powder of 1:2. 100 mL of deionized water was added to the mixed Polygonatum sibiricum powder, and the mixture was sealed and placed in a high-pressure steam sterilizer at 121℃ for 15 min for sterilization. The ratio of raw to cooked Polygonatum sibiricum added was 1:50, and the total amount was 2 g.

[0118] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, 2 mL (2%) of the bacterial concentration obtained in step (1) was added. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 25℃, 30℃, 35℃, 38℃ and 40℃ for 6 h respectively, and then aged at 4℃ for 12 h to obtain fermentation broth. The clarity, aroma and taste changes of the fermentation broth were evaluated respectively.

[0119] The sensory evaluation of the effect of fermentation temperature on the fermentation broth of Polygonatum odoratum is shown in Table 7.

[0120] Table 7: Sensory evaluation of fermentation temperature of fermentation broth

[0121]

[0122] The results showed that the fermentation bacteria functioned best at a fermentation temperature of 35℃, resulting in a clear fermentation liquid with a richer fermentation flavor, a stronger sweet aroma, no medicinal smell, and a weaker bitter taste, making it the most acceptable.

[0123] 5. The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0124] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they were mixed at a ratio of 1:2 between raw and cooked Polygonatum sibiricum powder. 100 mL of deionized water was added to each of the mixed Polygonatum sibiricum powders, and the mixture was sealed and placed in a high-pressure steam sterilizer at 121°C for 15 min for sterilization. The ratio of raw to cooked Polygonatum sibiricum powder was 1:50, and the total amount was 2 g.

[0125] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, 2 mL of the bacterial concentration obtained in step (1) was added to each solution. The bacterial concentration of each solution was 1×10⁻⁶. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 38℃ for 4 h, 6 h, 8 h, 10 h and 12 h respectively. Each treatment was aged at 4℃ for 12 h. The clarity, aroma and taste changes of the fermentation broth after 4 h, 6 h, 8 h, 10 h and 12 h were evaluated.

[0126] The sensory effects of fermentation time on the fermentation of compound Polygonatum powder are evaluated as shown in Table 8.

[0127] Table 8: Sensory evaluation of fermentation time of fermentation broth

[0128]

[0129] The results showed that the fermentation broth had the most intense flavor and a distinct sweet aroma at 8 hours; the fermentation flavor was weaker at 4 hours and 6 hours, with a medicinal smell and improved bitterness; and at 10 hours and 12 hours, it showed over-fermentation, off-flavors, increased turbidity of the fermentation broth, and a distinct sour and astringent taste.

[0130] Example 4: Analysis of active ingredients in fermented compound Polygonatum powder products

[0131] The specific steps are as follows:

[0132] The specific implementation method is the same as in Example 1, except that steps (2) and (3) are adjusted as follows:

[0133] (2) After processing raw and cooked Polygonatum sibiricum according to the processing method of Example 1, they are mixed in a ratio of raw Polygonatum sibiricum powder to cooked Polygonatum sibiricum powder of 1:2. 100 mL of deionized water is added to the mixed Polygonatum sibiricum powder, and the mixture is sealed and placed in a high-pressure steam sterilizer at 121℃ for 15 min for sterilization. The total amount of raw and cooked Polygonatum sibiricum added is 2 g.

[0134] (3) When the temperature of the high-pressure sterilized Polygonatum sibiricum solution was lowered to 30℃, 2 mL of the bacterial concentration obtained in step (1) was added to each solution. The bacterial concentration of each solution was 1×10⁻⁶. 7 Fermentation was carried out using CFU / mL Lactobacillus plantarum culture. Fermentation conditions: After sterilizing the above fermentation system with ultraviolet light for 30 min, fermentation was carried out at 38℃ for 8 h. After maturation at 4℃ for 12 h, the changes in several active ingredients such as polysaccharides, monosaccharides, short-chain fatty acids and saponins in the fermentation broth were further measured.

[0135] Meanwhile, the specific implementation method is the same as above, except that pure raw Polygonatum powder and pure cooked Polygonatum powder are used as comparative experiments; that is, 2g of raw Polygonatum powder or cooked Polygonatum powder is added directly.

[0136] (4) Result detection

[0137] 1) Polysaccharide content

[0138] The polysaccharide content extracted from the fermentation broth was determined using the sulfuric acid phenol total sugar determination method, and the results are as follows: Figure 1 As shown, the results are as follows:

[0139] Polysaccharides are the main functional components of Polygonatum sibiricum, and they have certain antioxidant, immunomodulatory, and anti-fatigue activities.

[0140] The initial polysaccharide content in the fermentation system using a mixture of raw and cooked Polygonatum was approximately 2.13 g / L. After 8 hours of fermentation with Lactobacillus plantarum, the polysaccharide content decreased to 1.38 g / L. The decrease in polysaccharide may be due to consumption by probiotics during fermentation or conversion into flavor or highly active substances.

[0141] The initial polysaccharide content in the fermentation system using raw Polygonatum powder was approximately 2.96 g / L, which decreased to 1.69 g / L after 8 hours of fermentation with Lactobacillus plantarum.

[0142] The initial polysaccharide content in the fermentation system using cooked Polygonatum powder was approximately 1.44 g / L, which decreased to 1.06 g / L after 8 hours of fermentation with Lactobacillus plantarum.

[0143] 2) Monosaccharide content:

[0144] The results are as follows Figure 2 As shown, the results are as follows:

[0145] Fermentation by Lactobacillus plantarum significantly affected the monosaccharide composition of Polygonatum sibiricum. Mannose, which has a sweet and bitter taste and is the main monosaccharide in Polygonatum sibiricum, was degraded in large quantities, decreasing from 0.85 g / L in unfermented Polygonatum sibiricum to 0.41 g / L in compound Polygonatum sibiricum.

[0146] Fermentation significantly enhances flavor, increasing the content of glucose, galactose, and other sugars. During fermentation, the content of most monosaccharides in Polygonatum decreases, while the content of easily absorbed monosaccharides—glucose, galactose, galacturonic acid, and arabinose—significantly increases. Compared to unfermented Polygonatum powder, the levels of glucose, galactose, galacturonic acid, and arabinose in fermented compound Polygonatum powder increased by 1.3, 1.2, 2.0, and 1.6 times, respectively. Compared to unfermented raw Polygonatum, fermented raw Polygonatum showed the same trend except for galactose, with glucose, galacturonic acid, and arabinose content increasing by 1.4, 1.2, and 1.9 times, respectively. Compared to unfermented cooked Polygonatum, fermented cooked Polygonatum showed increases of 1.2, 1.1, 1.7, and 2.1 times, respectively.

[0147] 3) Saponin Measurement

[0148] Saponins are irritating to the oral mucosa and are the main source of unpleasant sensory effects in Polygonatum sibiricum. Fermentation significantly reduces saponin content, as shown in the following results. Figure 3 As shown, the results are as follows:

[0149] The initial saponin content in the raw Polygonatum fermentation system was approximately 0.75 g / L. After 8 hours of fermentation with Lactobacillus plantarum, the saponin content decreased to 0.26 g / L; the saponin content decreased by 65% ​​after fermentation.

[0150] The initial saponin content in the fermentation system of cooked Polygonatum was about 0.15 g / L. After fermentation with Lactobacillus plantarum for 8 hours, the saponin content decreased to 0.07 g / L; the saponin content decreased by 53% after fermentation.

[0151] The initial saponin content in the compound Polygonatum fermentation system was approximately 0.36 g / L. After fermentation with Lactobacillus plantarum for 8 hours, the saponin content decreased to 0.09 g / L; the saponin content decreased by 76% after fermentation.

[0152] 4) Short-chain fatty acid content

[0153] Short-chain fatty acids play a role in maintaining gut health, regulating the immune system, regulating metabolism, protecting the cardiovascular system, and influencing brain health. Probiotics are the main bacterial flora that produce short-chain fatty acids. The results of the analysis of short-chain fatty acid content in fermented Polygonatum sibiricum are as follows... Figure 4 As shown in Table 9.

[0154] Table 9: Content of different short-chain fatty acids

[0155]

[0156]

[0157] Note: Unit is mg / L, - indicates not detected.

[0158] The results show:

[0159] The initial total amount of short-chain fatty acids in the raw Polygonatum fermentation system was approximately 87.02 mg / L. After fermentation with Lactobacillus plantarum for 8 hours, the content of polysaccharide saponins increased to 107.89 mg / L; the total amount increased by 1.24 times after fermentation.

[0160] The initial total amount of short-chain fatty acids in the fermentation system of cooked Polygonatum was about 15.85 mg / L. After fermentation with Lactobacillus plantarum for 8 hours, the total amount of short-chain fatty acids increased to 17.93 mg / L; the total amount increased by 1.13 times after fermentation.

[0161] The initial total amount of short-chain fatty acids in the compound Polygonatum fermentation system was approximately 71.15 mg / L. After 8 hours of fermentation with Lactobacillus plantarum, the total amount of short-chain fatty acids increased to 113.67 mg / L, representing a 1.60-fold increase.

[0162] These results demonstrate that the selected Lactobacillus plantarum can significantly enhance the sensory flavor of Polygonatum powder through fermentation. By selecting and blending raw and cooked Polygonatum, the nutritional components are balanced, and the irritation, bitterness, and strong medicinal odor of Polygonatum are reduced. The beneficial active ingredients of Polygonatum are significantly increased through blending and fermentation, which has great potential and application value for the preparation of beverages and foods using Polygonatum fermentation.

[0163] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. A fermentation method for compound Polygonatum, characterized in that, The method includes the following steps: (1) Mix raw and cooked Polygonatum sibiricum in a mass ratio of 1:2, add 100 mL of deionized water to the mixed Polygonatum sibiricum powder and sterilize; The ratio of Polygonatum sibiricum powder to deionized water is 1:50, g:mL; The raw Polygonatum is prepared by selecting fresh Polygonatum that has been pre-dried, washing and removing the rootlets, slicing it into pieces with a thickness of 2-5 mm, and then air-drying it to obtain raw Polygonatum slices with a moisture content of ≤6%. The prepared Polygonatum is prepared by: selecting fresh Polygonatum that has been pre-dried, steaming it in a steamer for 6 hours, turning off the heat and letting it sit for 8 hours, then taking it out and placing it in a 60 ℃ oven until the outer skin is slightly dry. This is the prepared Polygonatum prepared by steaming and drying once. The above method is repeated 9 times for steaming and drying, then cut and dried to obtain prepared Polygonatum prepared by nine steaming and nine drying, with a moisture content of ≤15%. (2) After adding microbial agents to the sterilized mixture and fermenting it, a compound Polygonatum fermentation liquid is obtained; The microbial agent is Lactobacillus plantarum bacterial solution; The amount of the microbial agent added is 2%; the concentration of the microbial agent is at least 1×10⁻⁶. 7 CFU / mL; The fermentation conditions were as follows: the fermentation system obtained by adding microbial agents to the sterilized mixture was sterilized by ultraviolet light for 20-30 minutes, fermented at 35℃ for 8 hours, and then matured at 4℃ for 12 hours.

2. The method according to claim 1, characterized in that, The method for preparing the Lactobacillus plantarum bacterial solution is as follows: (1) Inoculate Lactobacillus plantarum into M17 medium and culture at 35-37 ℃ to prepare Lactobacillus plantarum seed liquid; (2) The prepared Lactobacillus plantarum seed solution was inoculated into MRS broth medium at an inoculation rate of 1-10% and cultured at 35-37℃ and 150-200 r / min for 12-16 h to obtain the culture medium; (3) Centrifuge the obtained culture medium at 3000~5000 r / min and 2~4℃ for 5~10 min to collect the bacterial cells, and then dilute it with physiological saline to obtain Lactobacillus plantarum bacterial solution.

3. The method according to claim 2, characterized in that, Step (2) involves inoculating 1-10% of the prepared *Lactobacillus plantarum* seed culture into MRS broth and pre-culturing it at 37°C and 200 r / min for 16 h to obtain the culture medium. Step (3) involves centrifuging the culture medium at 5000 r / min and 4°C for 5 min to collect the bacterial cells, then diluting it with physiological saline to obtain a viable count of 10. 7 CFU / mL bacterial culture.

4. The compound Polygonatum fermentation broth prepared by any one of the methods described in claims 1 to 3.

5. A product containing the compound Polygonatum sibiricum fermentation liquid according to claim 4, wherein the product is a food, a drug, or a health product.

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