Huangjingegumi powder and a method for preparing the same

Abstract

The application discloses a kind of Huangjing Gouqi enzyme powder and the method for preparing it, belong to food processing technical field.The application provides a kind of Huangjing as main raw material, with black Gouqi using streptococcus thermophilus and lactobacillus mixed bacterial agent fermentation high-activity enzyme instant solid beverage powder preparation method of nine steaming nine sunning ripening.The method of the application, after probiotic fermentation, the quality of Huangjing enzyme is natural, flavor is prominent, functional ingredient content is high, polysaccharide content reaches 457.31mg / g, polyphenol content reaches 193.85mg / g, free amino acid content reaches 12.83mg / g, saponin content reaches 51.42mg / g, anthocyanin content reaches 96.18mg / g, and the content of health functional ingredients is significantly improved.

Description

Technical Field

[0001] This invention relates to a Polygonatum and Lycium barbarum enzyme powder and its preparation method, belonging to the field of food processing technology. Background Technology

[0002] Polygonatum sibiricum is a perennial herbaceous plant belonging to the genus Polygonatum in the family Liliaceae. Its rhizome, the main part used, is rich in active ingredients such as polysaccharides, saponins, flavonoids, and alkaloids, as well as starch, fat, protein, carotene, and vitamins. As a dual-purpose medicinal and edible resource in my country, Polygonatum can be used as food and has been proven to have antioxidant, anti-inflammatory, blood sugar and lipid-regulating, immune-enhancing, and tumor-suppressing effects. Compared to raw Polygonatum, which can irritate the throat and skin and cause numbness in the mouth and tongue, the nine-steaming and nine-drying process of cooked Polygonatum has a "reduced toxicity and enhanced efficacy" effect. Cooked Polygonatum has a mild, sweet taste and improved health benefits, including nourishing yin and lungs, lowering blood sugar, and enhancing immunity.

[0003] Black goji berries (Lycium ruthenicum Murray), also known as black wolfberries, are shrubs belonging to the Lycium genus of the Solanaceae family. They are rich in anthocyanins, as well as vitamins, polysaccharides, flavonoids, and minerals. Black goji berries possess strong antioxidant and anti-allergic activities, and can improve sleep, enhance vision, prevent cancer, and boost the immune system. They are considered to have higher nutritional value than regular goji berries.

[0004] Edible plant enzymes are enzyme products made from plants that can be used in food processing as the main raw material, with or without the addition of auxiliary materials, and are produced by microbial fermentation. They contain specific bioactive components and are suitable for human consumption.

[0005] The combination of Polygonatum and wolfberry is a classic and effective formula for the application of Polygonatum. It typically involves steaming peeled raw Polygonatum and regular wolfberry in a 1:1 ratio to extract the juice or making pills with honey. This method results in a strong medicinal flavor, a heavy bitter taste, unpleasant palatability, low content of effective functional components, complicated processing, and poor storage stability. To address these issues, a fermentation process is used, combining Polygonatum processed through nine steaming and nine sun-drying to enhance its efficacy with highly health-promoting black wolfberry to obtain an enzyme product. Fermentation reduces the medicinal odor, lessens the bitterness, and enhances the health-promoting functional components, resulting in a natural enzyme product that is easy to process, store, and consume.

[0006] Existing research on the preparation of Polygonatum sibiricum enzyme, for example (publication number CN 115486535A), discloses a method for preparing Polygonatum sibiricum enzyme: First, Polygonatum sibiricum, Lycium barbarum, and ginseng are mixed and slurried with water. The pH of the slurry is adjusted, and a compound enzyme is added for enzymatic hydrolysis to obtain an enzymatic hydrolysate. The enzymatic hydrolysate is then inactivated and fermented sequentially with yeast, lactic acid bacteria, and Streptococcus thermophilus. The resulting fermentation liquid is then sealed and chelated for fermentation. After filtration, Polygonatum sibiricum enzyme liquid is obtained. The Polygonatum sibiricum enzyme liquid is concentrated and dried to obtain Polygonatum sibiricum enzyme. However, this method has the following limitations: First, it directly uses raw Polygonatum sibiricum, which has a strong irritant taste and affects sensory quality. Second, it may be limited by the low concentration of added bacterial solution and the long fermentation time, with aerobic fermentation taking 6-9 days and room temperature chelation fermentation taking 40-50 days. Third, the drying method involves prolonged high-temperature drying, which leads to the loss of nutrients and flavor components. China has filed a patent application (CN114009778 A) for a method to prepare a highly active Polygonatum sibiricum instant powder. The method involves selecting Polygonatum sibiricum, removing the roots, and cleaning it. After moistening, the powder undergoes nine steaming and nine processing cycles. The processed Polygonatum sibiricum is then sliced, pulverized, sieved, flash-extracted, filtered, and dried using microwave vacuum after adding excipients. Its limitations are: first, while the efficacy of Polygonatum sibiricum is enhanced through nine steaming and nine processing cycles, the active ingredients, such as polysaccharides, remain primarily in large, poorly absorbed molecular forms; second, the processed Polygonatum sibiricum has a strong medicinal flavor and poor sensory quality; third, the large amount of maltodextrin added as an excipient leads to a decrease in the content of Polygonatum sibiricum, and is also unsuitable for certain populations, such as diabetics.

[0007] In existing technologies, there is limited research on Polygonatum enzymes and instant beverages. These primarily use raw Polygonatum, resulting in complex and time-consuming production processes, numerous byproducts, and difficulty in quality control. The products do not consider enhancing sensory flavor, and the lack of removal of insoluble solids further complicates convenience. Instant Polygonatum products often only involve extraction without utilizing probiotic fermentation to enhance functional components and sensory flavor. Furthermore, probiotic fermentation often lacks strain selection and optimization. Compared to the closest similar solutions, which involve cumbersome raw material processing, complex ingredient formulations, and are unsuitable for large-scale production, and whose excessive additives may be unsuitable for certain populations, this invention offers advantages such as simple raw material processing, mild processing conditions, short processing time, low environmental and equipment requirements, no exogenous additives, high content of effective functional components from Polygonatum, naturally suitable product quality for a wide range of consumers, and the potential for large-scale production. Summary of the Invention

[0008] To address the aforementioned issues, this invention utilizes highly effective, nine-steamed and nine-dried processed Polygonatum sibiricum with selected probiotics, combined with black goji berries, to produce an instant enzyme solid beverage through fermentation. This avoids the numbing sensation and other irritations associated with raw Polygonatum sibiricum, while further enzymatic hydrolysis increases the content of polysaccharides and other nutrients. The co-fermentation with highly nutritious black goji berries significantly enhances beneficial components such as polyphenols (anthocyanins, flavonoids, etc.), vitamins, and trace elements, further breaking down large nutrient molecules into smaller, more easily digestible molecules. Natural osmanthus honey is added to promote fermentation and enhance flavor. Considering market demand, after removing coarse residue, vacuum freeze-drying technology is used to obtain the instant enzyme powder, maintaining nutritional value and flavor while reducing the use of additives. This invention is simple to operate, convenient and quick to produce, and easy to promote and prepare.

[0009] This invention provides a method for preparing a highly active enzyme instant solid beverage powder using nine-steamed and nine-dried processed Polygonatum as the main raw material, combined with black wolfberry fermented using a mixed inoculum of Streptococcus thermophilus and Lactobacillus. The method should also include Polygonatum enzyme solution, concentrated slurry, etc.

[0010] This invention provides a method for preparing a compound enzyme of Polygonatum sibiricum and black goji berries, the method comprising the following steps:

[0011] (1) After mixing cooked Polygonatum and black wolfberry in a mass ratio of 2:1 to 1:1, add water to soak them in a material-to-liquid ratio of 1:10 (g / mL), and then homogenize them using a tissue homogenizer to obtain a homogenate.

[0012] (2) Add honey to the homogenate obtained in step (1) at a mass fraction of 4-6%, and add a mixture of cellulase and pectinase. After mixing, enzymatically hydrolyze at 40-50℃ for 2-4 hours. The enzyme activity of the added cellulase is 50-100 U / mg; the enzyme activity of the added pectinase is 400-800 U / mg. Obtain the enzymatic hydrolysate, sterilize it and set it aside for later use.

[0013] (3) Inoculate the fermentation agent into the sterilized enzymatic hydrolysate obtained in step (2) and ferment at 36-38℃ for 48-72h; respectively prepare Polygonatum compound black goji berry enzyme; the fermentation agent is Streptococcus thermophilus liquid, Lactobacillus plantarum liquid and / or Lactobacillus casei liquid.

[0014] In one embodiment of the present invention, the cooked Polygonatum is a high-quality Polygonatum that has been steamed and sun-dried nine times.

[0015] In one embodiment of the present invention, in step (2), the cellulase and pectinase mixture is added in a mass ratio of 1:1.

[0016] In one embodiment of the present invention, the amount of the cellulase and pectinase mixture enzyme preparation added is 5% of the total amount of Polygonatum and Lycium barbarum.

[0017] In one embodiment of the present invention, the cellulase has an added enzyme activity of 50 U / mg; the pectinase has added enzyme activity of 500 U / mg; the enzymatic hydrolysate is obtained by enzymatic hydrolysis for 2 hours at a temperature of 50°C, and the enzymatic hydrolysate is sterilized and then cooled.

[0018] In one embodiment of the present invention, in step (2), the honey includes, but is not limited to, osmanthus honey, milkvetch honey, lychee honey, and multifloral honey.

[0019] In one embodiment of the present invention, in step (3), the number of viable bacteria in the fermentation agent is at least 10. 8 CFU / mL.

[0020] In one embodiment of the present invention, in step (3), the fermentation agent is a fermentation agent obtained by mixing Streptococcus thermophilus liquid, Lactobacillus plantarum liquid and Lactobacillus casei liquid in a volume ratio of 1:(1-2):1.

[0021] In one embodiment of the present invention, the fermentation agent is inoculated into the sterilized enzymatic hydrolysate at a volume ratio of 4 to 6%.

[0022] In one embodiment of the present invention, the preparation method of the Streptococcus thermophilus bacterial solution, Lactobacillus plantarum bacterial solution, and Lactobacillus casei bacterial solution is as follows:

[0023] (1) Inoculate Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus casei into MRS broth at an inoculation rate of 1-10% and pre-culture at 35-37℃ and 150-300r / min for 12-16h as seed culture.

[0024] (2) After centrifuging the *Streptococcus thermophilus* seed solution, *Lactobacillus plantarum* seed solution, and *Lactobacillus casei* seed solution obtained in step (1), collect the bacterial cells, and then dilute them with physiological saline to obtain viable bacterial counts of 10⁻⁶. 8 CFU / mL of Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus casei.

[0025] In one embodiment of the present invention, the preparation method of the Streptococcus thermophilus bacterial solution, Lactobacillus plantarum bacterial solution, and Lactobacillus casei bacterial solution is as follows:

[0026] Streptococcus thermophilus, Lactobacillus plantarum and Lactobacillus casei were inoculated into MRS broth at an inoculation rate of 1-10% and pre-cultured at 37℃ and 200r / min for 16h as seed culture.

[0027] The above pre-cultured seed culture solution was centrifuged at 5000 r / min and 4℃ for 5 min to collect bacterial cells. After dilution with physiological saline, the viable cell count was approximately 10. 8 CFU / mL single-strain bacterial culture.

[0028] In one embodiment of the present invention, the sterilization method of the enzymatic hydrolysate in step (2) is to perform high-pressure sterilization at 121°C for 10 to 20 minutes.

[0029] The present invention also provides a Polygonatum odoratum compound black goji berry enzyme prepared by the method.

[0030] The present invention also provides an enzyme freeze-dried powder containing the above-mentioned Polygonatum odoratum compound black goji berry enzyme. The freeze-dried powder is obtained by filtering the enzyme liquid prepared by the above method to remove coarse residue, concentrating, freeze-drying and pulverizing to obtain the enzyme freeze-dried powder.

[0031] In one embodiment of the present invention, the filtration freeze-drying is performed by: diluting the obtained fermented enzyme liquid multiple times to remove coarse residue, collecting the suspension and concentrating it to obtain concentrated original pulp, then performing vacuum freeze-drying, pulverizing the freeze-dried material, and obtaining a Polygonatum and Lycium barbarum enzyme instant solid beverage.

[0032] The present invention also provides a product containing the above-mentioned Polygonatum odoratum compound black goji berry enzyme, the product being a food or health product.

[0033] This invention provides a compound enzyme liquid of Polygonatum and Lycium barbarum, an enzyme concentrate, and an instant enzyme solid beverage prepared by the above method.

[0034] This invention provides a method for preparing enzyme products using cooked Polygonatum sibiricum (steamed and sun-dried nine times) combined with black goji berries. The method uses cooked Polygonatum sibiricum and black goji berries as raw materials and prepares enzyme food through probiotic fermentation.

[0035] Beneficial effects

[0036] This invention provides a method for preparing an enzyme product using a combination of nine-steamed and nine-dried processed Polygonatum sibiricum and black goji berries. Both the processed Polygonatum sibiricum and black goji berries are raw materials with high health benefits. Homogenization and enzymatic hydrolysis promote the degradation of poorly absorbed pectin and fiber, thereby increasing the content of beneficial polysaccharides in the raw material matrix. Natural honey is added to enhance the flavor and provide a suitable carbon source for probiotic growth, while also promoting the dissolution of polyphenols and other components. Further optimization of probiotics for short-term fermentation avoids the production of high ethanol content and further degrades large-molecule polysaccharides into easily absorbed small-molecule sugars, significantly increasing the content of health-promoting components. The pungent saponins and bitterness in Polygonatum sibiricum are significantly reduced, the unique medicinal odor of processed Polygonatum sibiricum is removed, and a distinct sweet aroma from fermentation is achieved. The Polygonatum enzyme obtained through probiotic fermentation is of natural quality, has a prominent flavor, and a high content of functional components. The polysaccharide content reaches 457.31 mg / g, the polyphenol content reaches 193.85 mg / g, the free amino acid content reaches 12.83 mg / g, the saponin content reaches 51.42 mg / g, and the anthocyanin content reaches 96.18 mg / g, with a significant increase in the content of health-promoting functional components. Attached Figure Description

[0037] Figure 1 The process flow for preparing Polygonatum and Lycium barbarum compound enzyme according to the method of the present invention is as follows.

[0038] Figure 2 The method of this invention is used to prepare Polygonatum and Lycium barbarum compound enzyme powder.

[0039] Figure 3 The method of this invention prepares the Polygonatum and Lycium barbarum compound enzyme raw material and brewing liquid.

[0040] Figure 4 The polysaccharide content of the Polygonatum and Lycium barbarum compound enzyme powder prepared by the method of this invention.

[0041] Figure 5 The polyphenol content of the Polygonatum and Lycium barbarum compound enzyme powder prepared by the method of this invention. Detailed Implementation

[0042] Both Polygonatum and Lycium barbarum are rich in polysaccharides, making them excellent fermentation substrates for probiotics. Probiotic fermentation can promote the further degradation of poorly absorbed macromolecules. However, due to differences in the metabolic capacity and pathways of the fermenting agents, the overall aroma, taste, color, flavor, and sensory quality of fermented products mainly depend on the type of fermenting agent. Mixed probiotics, compared to single strains, metabolize prebiotics in greater quantity and variety, effectively enhancing nutritional components and promoting the growth of different strains. Streptococcus thermophilus and Lactobacillus are commonly used food fermenting agents, widely applied in improving the nutritional quality and flavor of food. Cooked Polygonatum has a strong medicinal aroma and bitter taste; combining it with black Lycium barbarum and honey increases nutritional components and harmonizes the flavor. Further fermentation with mixed agents improves the flavor and nutritional components of Polygonatum products, better meeting people's demand for natural health benefits from Polygonatum.

[0043] The sensory evaluation of enzymes was conducted in accordance with the indicators for edible plant enzymes in QB / T 5323-2018 "Plant Enzymes", as shown in Table 1.

[0044] Table 1: Sensory Evaluation Criteria for Compound Enzymes

[0045]

[0046] All raw materials used in the following examples are commercially available. The nine-steamed and nine-dried processed Polygonatum was sourced from Zhejiang Senyu Industrial Co., Ltd., and the dried black goji berries were purchased from Ningxia Qilixiang Goji Berry Co., Ltd. The food-grade fermentation agent used in the following examples was purchased from Beijing Chuanxiu Technology Co., Ltd.

[0047] The *Streptococcus thermophilus*, *Lactobacillus plantarum*, and *Lactobacillus casei* strains involved in the following embodiments 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*. In this invention, for ease of description, *Streptococcus thermophilus*, *Lactobacillus plantarum*, and *Lactobacillus casei* are respectively named *Streptococcus thermophilus* IMAU, *Lactobacillus plantarum* NH, and *Lactobacillus casei* 4N. Furthermore, the applicant undertakes to release the aforementioned biological materials—*Streptococcus thermophilus* IMAU, *Lactobacillus plantarum* NH, and *Lactobacillus casei* 4N—to the public within twenty years from the date of application.

[0048] The culture media involved in the following examples are as follows:

[0049] M17 medium (product code: PM0710, g / L): soybean peptone 5.0, peptone 5.0, 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. Dissolve 42.3g in 1L of distilled water by heating, and autoclave at 121℃ for 15min.

[0050] 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. Dissolve 54g in 1L of distilled water, stir and heat until dissolved, then autoclave at 121℃ for 15min.

[0051] The detection methods involved in the following embodiments are as follows:

[0052] Characteristic indicators were determined for the optimized enzyme preparation from Polygonatum and Lycium barbarum: including general physicochemical indicators (pH) and characteristic physicochemical indicators (total amount of crude polysaccharides, polyphenols, and free amino acids), and the contents of saponins, a characteristic component of Polygonatum, and anthocyanins, a characteristic component of Lycium barbarum, were also detected.

[0053] (1) pH determination: Take 0.5g of lyophilized enzyme sample, add 10mL of distilled water, sonicate for 5min to mix thoroughly, and then centrifuge at 8000r / min to collect the supernatant. Use a calibrated pH meter to determine the pH of the supernatant.

[0054] (2) Determination of crude polysaccharide content: Take 5g of lyophilized enzyme sample, add 10mL of distilled water and extract at 55℃ for 6h, add 30mL of anhydrous ethanol and place in a 4℃ refrigerator for precipitation for 24h, centrifuge at 8000r / min for 10min, remove the solution and retain the precipitate; repeat the ethanol precipitation step to obtain crude polysaccharide of Polygonatum and Lycium barbarum enzyme. The polysaccharide content extracted from the fermentation broth was determined by the total sugar determination method of phenol sulfate.

[0055] (3) Polyphenol content determination: Take 0.5g of enzyme freeze-dried sample into a 25mL stoppered test tube, add 10mL of 60% ethanol solution, sonicate for 10min, extract in a 75℃ water bath for 1h, centrifuge at 8000r / min for 10min and take 1mL of supernatant, and determine the polyphenol content using the Folin-phenol colorimetric method.

[0056] (4) Determination of total free amino acids: Weigh 1g of lyophilized enzyme sample, add 5.0mL of 10% acetic acid solution, and dilute to volume with distilled water in a 100mL volumetric flask. Centrifuge at 8000r / min for 10min, take 1mL of supernatant, and determine the total free amino acids using the ninhydrin colorimetric method.

[0057] (5) Determination of saponin content: Take 1g of lyophilized enzyme sample, add 80% ethanol, bring the volume to 50mL at 70℃, and reflux for 2h. Repeat the extraction twice, centrifuge at 8000r / min to collect the supernatant, concentrate by rotary evaporation, and extract three times with n-butanol at a 1:1 ratio. Then concentrate under reduced pressure to obtain crude extract of Polygonatum saponins. Take 0.2g of crude saponin extract, bring the volume to 100mL with methanol, and use ginsenoside Rb1 as standard to determine the saponin content according to the vanillin-perchloric acid colorimetric method.

[0058] (6) Anthocyanin content determination: Take 0.5g of enzyme freeze-dried sample and place it in a 150mL stoppered conical flask. Add 50mL of 3% hydrochloric acid ethanol solution, extract by ultrasonication at 50℃ for 30min, cool to room temperature, centrifuge at 8000r / min for 10min, take 1mL of supernatant and determine the total anthocyanin content by pH differential method.

[0059] Example 1: Material Preparation

[0060] (1) Preparation of Polygonatum and Lycium chinense homogenate

[0061] Selected processed Polygonatum sibiricum and destemmed black wolfberry dried products were prepared by steaming and sun-drying nine times. After soaking Polygonatum sibiricum and wolfberry at a ratio of 1:10 (g / mL), they were homogenized using a tissue homogenizer.

[0062] The process of nine-steaming and nine-drying processed Polygonatum is as follows: Selected fresh Polygonatum that has been pre-dried is steamed for 6 hours, then left to moisten for 8 hours after the heat is turned off. It is then placed in a 60℃ oven until the outer skin is slightly dry, resulting in one-time steaming and drying of Polygonatum. This process is repeated nine times, followed by slicing and drying, to obtain nine-steaming and nine-drying processed Polygonatum with a moisture content ≤15%. The sliced ​​processed Polygonatum is then steeped in boiling water to obtain the steeping liquid (…). Figure 3 ).

[0063] (2) Preparation of seed liquid

[0064] Streptococcus thermophilus IMAU, Lactobacillus plantarum NH, and Lactobacillus casei 4N strains were inoculated into MRS broth at inoculation rates of 1–10% and pre-cultured at 37°C and 200 r / min for 16 h to prepare Streptococcus thermophilus seed culture, Lactobacillus plantarum seed culture, and Lactobacillus casei seed culture, respectively.

[0065] (3) Preparation of mixed bacterial agent

[0066] The above-mentioned *Streptococcus thermophilus* IMAU seed culture, *Lactobacillus plantarum* NH seed culture, and *Lactobacillus casei* 4N seed culture were centrifuged at 5000 r / min and 4℃ for 5 min, respectively, to collect bacterial cells. After dilution with physiological saline, the viable count of a single strain was approximately 10. 8 CFU / mL of pure bacterial cultures with different strains:

[0067] The number of live bacteria is approximately 10. 8 The cFU / mL Streptococcus thermophilus IMAU bacterial suspension has a viable count of approximately 10. 8 The CFU / mL Lactobacillus plantarum NH bacterial suspension and viable count were approximately 10. 8 CFU / mL Lactobacillus casei 4N bacterial suspension.

[0068] The fermentation inoculant is prepared as follows:

[0069] Group (A): Streptococcus thermophilus IMAU bacterial suspension, viable count: 10 8 CFU / mL;

[0070] Group (B): Lactobacillus plantarum NH bacterial suspension, viable count: 10 8 CFU / mL;

[0071] Group (C): Lactobacillus casei 4N bacterial suspension, viable count: 10-1 8 CFU / mL;

[0072] The thermophilic streptococcus bacterial suspension, lactobacillus plantarum bacterial suspension, and lactobacillus casei bacterial suspension obtained from the above steps can be mixed in different volume ratios to form a mixed bacterial agent.

[0073] Group (D): The fermentation inoculum was obtained by mixing *Streptococcus thermophilus* IMAU culture and *Lactobacillus plantarum* NH culture (volume ratio: v / v = 1:1), with a viable cell count of approximately 10. 8 CFU / mL;

[0074] Group (E): The fermentation inoculum was obtained by mixing *Streptococcus thermophilus* IMAU culture and *Lactobacillus casei* 4N culture (volume ratio: v / v = 1:1), with a viable cell count of approximately 10. 8 CFU / mL;

[0075] Group (F): The fermentation inoculum was obtained by mixing *Lactobacillus plantarum* NH bacterial suspension and *Lactobacillus casei* 4N bacterial suspension (volume ratio: v / v = 1:1), with a viable cell count of approximately 10. 8 CFU / mL;

[0076] Group (G): A fermentation inoculum was obtained by mixing *Streptococcus thermophilus* IMAU culture, *Lactobacillus plantarum* NH culture, and *Lactobacillus casei* 4N culture (volume ratio, v:v:v = 1:(1-2):1), with a viable cell count of approximately 10. 8 CFU / mL.

[0077] Example 2: Optimization of preparation conditions for Polygonatum and Lycium barbarum enzyme solution

[0078] The processing flow of the compound enzyme preparation of Polygonatum and Lycium barbarum according to the method of the present invention is as follows: Figure 1 As shown, the specific steps are as follows:

[0079] 1. Screening of fermentation inoculants

[0080] (1) Prepare the fermentation agent

[0081] Example 1: Preparation of fermentation inoculum: Groups (A) to (G);

[0082] Meanwhile, a commercially available enzyme fermentation powder was used as a control: (H) Group: Commercially available fruit and vegetable enzyme fermentation agent I (purchased from Beijing Chuanxiu Technology Co., Ltd., containing Lactobacillus plantarum, Lactobacillus acidophilus, Lactobacillus paracasei, Bifidobacterium lactis, and Lactobacillus rhamnosus); The treatment method was as follows: 0.1g of fermentation powder was added to an appropriate amount of sterile water and reactivated at 37℃, then diluted to adjust the viable count to approximately 10. 8 CFU / mL;

[0083] Group (I): Commercially available fruit and vegetable enzyme fermentation agent II (purchased from Angel Yeast Co., Ltd., containing Lactobacillus plantarum N13, Lactobacillus bulgaricus, Lactobacillus helveticus, Leuconostoc mesenteroides subsp. enterica, and Streptococcus thermophilus); the processing method is as follows: take 0.1g of fermentation powder, add an appropriate amount of sterile water, revive at 37℃, and dilute to adjust the viable count to approximately 10. 8 CFU / mL.

[0084] (2) The dried Polygonatum and destemmed black wolfberry were steamed and sun-dried nine times; Polygonatum and wolfberry (Polygonatum and wolfberry were mixed in a mass ratio of 1:1) were soaked in water at a ratio of 1:10 (g / mL), and then homogenized using a tissue homogenizer to obtain a homogenate.

[0085] (3) Add 4% (v / v) osmanthus honey to the homogenate obtained in step (2), and add a mixture of cellulase and pectinase (m / m = 1:1) at a mass fraction of 5%. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme activity of the added cellulase is 50 U / mg; the enzyme activity of the added pectinase is 500 U / mg; and the enzymatic hydrolysate is obtained.

[0086] (4) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0087] (5) Inoculate each group of fermentation agents from step (1) into the sterilized enzymatic hydrolysate obtained in step (4) at an inoculation rate of 4% (v / v) and ferment at 37°C for 48 hours to obtain enzyme solutions.

[0088] (6) Filter the enzyme liquid obtained in step (5) to remove coarse residue, concentrate, freeze dry, and pulverize to obtain enzyme freeze-dried powder.

[0089] Based on sensory evaluation, a superior probiotic fermentation agent for preparing enzymes from Polygonatum odoratum and black goji berries that have undergone nine steaming and nine sun-drying processes was selected, and the results are shown in Table 2.

[0090] Table 2: Sensory evaluation of enzymes prepared with different starter cultures

[0091]

[0092]

[0093] Sensory evaluation results show that:

[0094] (1) Comparing different fermentation groups, the flavor enhancement brought by mixed inoculum fermentation was significantly better than that of single inoculum fermentation;

[0095] (2) The effect of mixed bacterial agents on improving the flavor quality of Polygonatum and Lycium barbarum enzymes showed that: the mixture of two purified strains < commercial fruit and vegetable enzyme fermentation agents < the mixture of three purified strains.

[0096] (3) The enzyme obtained by fermentation of Streptococcus thermophilus has a uniform and moderate color, and the brewing liquid is clear and bright; Lactobacillus plantarum has the most obvious sensory effect in enhancing the taste and aroma of enzymes; Lactobacillus casei can improve the texture of enzyme powder.

[0097] By mixing three probiotic fermentation agents, the concentration and strains are more suitable for the application of the nine-steaming and nine-drying process of Polygonatum odoratum and black goji berry enzyme compared with commercial fermentation agents. The resulting enzyme product has a uniform and moderate color, dissolves quickly and is easy to brew; the taste is harmonious and mellow, sweet and sour and refreshing, without any numbing or astringent feeling; the sugary sweetness, fruity aroma and floral aroma are harmonious, the fermentation aroma is rich, and the acceptance level is the highest.

[0098] Therefore, based on comprehensive sensory evaluation, a mixed inoculum of Streptococcus thermophilus, Lactobacillus plantarum, and Lactobacillus casei was selected as the microbial fermentation agent for Polygonatum and Lycium barbarum enzyme products.

[0099] 2. Freeze-dried powders obtained from different weight ratios of Polygonatum and Lycium barbarum

[0100] (1) Selected dried Polygonatum sibiricum and destemmed black wolfberry: Polygonatum sibiricum and wolfberry were mixed in mass ratios of 5:1, 2:1 and 1:1 respectively, and the material-liquid ratio was fixed at 1:10 (g / mL). After soaking in water, the mixture was homogenized using a tissue homogenizer.

[0101] (2) Add 4% (v / v) osmanthus honey to the homogenate obtained in step (1), and add a mixture of cellulase and pectinase (m / m = 1:1) at a mass fraction of 5%. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The added cellulase has an enzyme activity of 50 U / mg, and the added pectinase has an enzyme activity of 500 U / mg. The enzymatic hydrolysate is obtained.

[0102] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0103] (4) The fermentation agent of group (G) in step 1, namely Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v = 1:(1-2):1), is mixed and inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at an inoculation amount of 4% (volume ratio: v / v), and fermented at 37°C for 48 hours to prepare the enzyme liquid.

[0104] (5) Filter the enzyme liquid obtained in step (4) to remove coarse residue, concentrate, freeze dry, and pulverize to obtain enzyme freeze-dried powder.

[0105] The effects of different ratios of Polygonatum and Lycium barbarum on the quality of the compound enzyme were investigated, and the results are shown in Table 3.

[0106] 3. Freeze-dried powders obtained with different amounts of honey added

[0107] (1) Mix Polygonatum and Lycium barbarum in a mass ratio of 2:1, add water to soak them at a fixed material-to-liquid ratio of 1:10 (g / mL), and homogenize them using a tissue homogenizer to obtain a homogenate.

[0108] (2) Add 2%, 4%, and 6% (v / v) osmanthus honey to the homogenate obtained in step (1), and further add 5% enzyme preparation according to the mass ratio. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme preparation is cellulase and pectinase (mass ratio: m / m = 1:1). The enzyme activity of the added cellulase is 50 U / mg, and the enzyme activity of the added pectinase is 500 U / mg. The enzymatic hydrolysate is obtained.

[0109] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0110] (4) The fermentation agent of group (G) in step 1, namely Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v = 1:(1-2):1), is mixed and inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at an inoculation amount of 4% (volume ratio: v / v), and fermented at 37°C for 48 hours to prepare the enzyme liquid.

[0111] (5) Filter the enzyme liquid obtained in step (4) to remove coarse residue, concentrate, freeze dry, and pulverize to obtain enzyme freeze-dried powder.

[0112] The effects of different amounts of honey added on the quality of the compound enzyme were investigated, and the results are shown in Table 3.

[0113] 4. Freeze-dried powders obtained with different amounts of mixed fermentation inoculants

[0114] (1) Mix Polygonatum and Lycium barbarum in a mass ratio of 2:1, add water to soak them at a fixed material-to-liquid ratio of 1:10 (g / mL), and homogenize them using a tissue homogenizer to obtain a homogenate.

[0115] (2) Add 4% (v / v) osmanthus honey to the homogenate obtained in step (1), and further add 5% enzyme preparation according to the mass ratio. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme preparation is cellulase and pectinase (mass ratio: m / m = 1:1). The enzyme activity of the added cellulase is 50 U / mg. The enzyme activity of the added pectinase is 500 U / mg. The enzymatic hydrolysate is obtained.

[0116] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0117] (4) The fermentation agent of group (G) in step 1, namely, the mixed agent of Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v = 1:(1-2):1), is inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at inoculation amounts of 2%, 4% and 6% (volume ratio: v / v), and fermented at 37°C for 48 hours to prepare the enzyme solution.

[0118] (5) Filter the enzyme liquid obtained in step (4) to remove coarse residue, concentrate, freeze dry and pulverize to obtain enzyme freeze-dried powder.

[0119] The effects of different mixed fermentation agents on the quality of the compound enzyme were investigated, and the results are shown in Figure 3.

[0120] 5. Freeze-dried powders obtained at different fermentation temperatures

[0121] (1) Mix Polygonatum and Lycium barbarum in a mass ratio of 2:1, add water to soak them at a fixed material-to-liquid ratio of 1:10 (g / mL), and homogenize them using a tissue homogenizer to obtain a homogenate.

[0122] (2) Add 4% (v / v) osmanthus honey to the homogenate obtained in step (1), and further add 5% enzyme preparation according to the mass ratio. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme preparation is cellulase and pectinase (mass ratio: m / m = 1:1). The enzyme activity of the added cellulase is 50 U / mg. The enzyme activity of the added pectinase is 500 U / mg. The enzymatic hydrolysate is obtained.

[0123] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0124] (4) The fermentation agent of group (G) in step 1, namely the mixed agent of Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v=1:(1-2):1), is inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at an inoculation amount of 4% (volume ratio: v / v), and fermented at fermentation temperatures of 32℃, 36℃ and 38℃ for 48h respectively; to prepare enzyme liquid.

[0125] (5) Filter the enzyme liquid obtained in step (4) to remove coarse residue, concentrate, freeze dry and pulverize to obtain enzyme freeze-dried powder.

[0126] The effects of different fermentation temperatures on the quality of the compound enzyme were investigated, and the results are shown in Table 3.

[0127] 6. Freeze-dried powders obtained for different fermentation times

[0128] (1) Mix Polygonatum and Lycium barbarum in a mass ratio of 2:1, add water to soak them at a fixed material-to-liquid ratio of 1:10 (g / mL), and homogenize them using a tissue homogenizer to obtain a homogenate.

[0129] (2) Add 4% (v / v) osmanthus honey to the homogenate obtained in step (1), and further add 5% enzyme preparation according to the mass ratio. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme preparation is cellulase and pectinase (mass ratio: m / m = 1:1). The enzyme activity of the added cellulase is 50 U / mg. The enzyme activity of the added pectinase is 500 U / mg. The enzymatic hydrolysate is obtained.

[0130] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0131] (4) The fermentation agent of group (G) in step 1, namely the mixed agent of Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v=1:(1-2):1), was inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at an inoculation amount of 4% (volume ratio: v / v), and fermented at 37℃ for 12h, 48h and 72h respectively; enzyme solutions were prepared respectively.

[0132] (5) The compound enzyme liquid obtained in step (4) is filtered to remove coarse residue, concentrated, freeze-dried and pulverized to obtain enzyme freeze-dried powder.

[0133] The effects of different fermentation times on the quality of the compound enzyme were investigated, and the results are shown in Table 3.

[0134] Table 3: Sensory evaluation of different optimized indicators of Polygonatum and Lycium barbarum compound enzyme

[0135]

[0136]

[0137] The results showed that the medicinal aroma of Polygonatum was significantly enhanced after nine steaming and nine sun-drying processes, and the bitterness and astringency of the taste became more pronounced after the initial sweetness and mellowness. Black goji berries are rich in anthocyanins and mainly have a bitter and astringent taste. Both are rich in polysaccharides, and their flavor is enhanced after fermentation. Comparing different weight ratios, when the content of cooked Polygonatum was much higher than that of black goji berries, the color was mainly dark brown, dominated by cooked Polygonatum. As the ratio of Polygonatum to black goji berries decreased, the pH decreased after fermentation, and the color turned into a bright red. The difference in tissue morphology was not significant, and the main differences were in taste and aroma. As the ratio of Polygonatum decreased, the bitterness and astringency decreased, the fermented taste became sweet and sour, and the fermented aroma became rich in floral and fruity fragrance.

[0138] (1) Osmanthus honey is preferred because it has a rich floral aroma and is rich in sugar, which can effectively enhance the flavor. When the amount added is 4%, it retains the flavor of Polygonatum odoratum while having a sweet and sour taste, a pleasant sweet and floral aroma, and suppresses the medicinal smell of cooked Polygonatum odoratum.

[0139] (2) A suitable inoculation amount facilitates substrate fermentation and promotes its own proliferation. With the increase of the inoculation amount, the flavor changes significantly at the same time. With an inoculation amount of 4%, the bitterness is reduced, the sweet and sour taste is moderate, and the sweet and fermented aroma is obvious. With an inoculation amount of 6%, the fermented aroma is sufficient, and the taste is slightly sour.

[0140] (3) Temperature is an important indicator affecting probiotic fermentation. At the same time, a suitable temperature is also conducive to the degradation of the components of Polygonatum and Lycium barbarum. When fermented at 38℃, the bitterness of the enzyme is significantly inhibited, the astringency is reduced, the taste is sweet and sour, and the fermentation aroma is obvious while highlighting the sweet aroma of cooked Polygonatum.

[0141] (4) Under semi-solid substrate fermentation conditions, the fermentation effect of the fermentation bacteria is best after 48 hours. After 72 hours, the fermentation aroma is rich and the sugar aroma is obvious. While retaining the flavor of Polygonatum and wolfberry, it is also sweet and sour.

[0142] 7. The optimized preparation process for Polygonatum and Lycium barbarum enzyme powder is as follows:

[0143] (1) Mix Polygonatum and Lycium barbarum in a mass ratio of 2:1, add water to soak them at a fixed material-to-liquid ratio of 1:10 (g / mL), and homogenize them using a tissue homogenizer to obtain a homogenate.

[0144] (2) Add 4-6% (v / v) of osmanthus honey to the homogenate obtained in step (1), and further add 5% of enzyme preparation according to the mass ratio. After mixing, enzymatically hydrolyze at 50°C for 2 hours. The enzyme preparation is cellulase and pectinase (mass ratio: m / m = 1:1). The enzyme activity of the added cellulase is 50 U / mg. The enzyme activity of the added pectinase is 500 U / mg. The enzymatic hydrolysate is obtained.

[0145] (3) Sterilize the enzymatic hydrolysate at 121°C for 10-20 minutes.

[0146] (4) The fermentation agent (G) from step 1, namely the mixed agent of Streptococcus thermophilus + Lactobacillus plantarum + Lactobacillus casei (volume ratio, v:v:v = 1:(1-2):1), is inoculated into the sterilized enzymatic hydrolysate obtained in step (3) at an inoculation rate of 4-6% (volume ratio: v / v), and fermented at 38°C for 48-72 hours; an enzyme solution (i.e., Figure 3 (The original pulp of nine-steamed and nine-dried Solomon's Seal + black goji berry enzyme).

[0147] (5) Filter the enzyme solution obtained in step (4) to remove coarse residue, concentrate, freeze-dry, and pulverize to obtain freeze-dried enzyme powder. Figure 2 The aqueous solution obtained by dissolving the lyophilized enzyme powder in water is as follows: Figure 3 The image shows a brewing liquid made from nine-steamed and nine-dried Solomon's Seal and black goji berry enzymes.

[0148] Example 3: Determination of characteristic indicators of Polygonatum and Lycium barbarum enzymes

[0149] The characteristic index analysis of the enzyme obtained by the optimized preparation process of Polygonatum and Lycium barbarum enzyme powder in step 7 of Example 2 is shown in Table 4.

[0150] Table 4: Results of determination of characteristic indicators of Polygonatum and Lycium barbarum compound enzyme

[0151]

[0152] Note: The ratio of nine-steamed and nine-dried Polygonatum to black wolfberry is 2:1; general physicochemical indicators should also include ethanol content (g / 100g). The freeze-dried enzymes do not contain ethanol and meet the indicator requirements.

[0153] The results show:

[0154] (1) A pH value ≤ 4.5 is a basic requirement for the general physicochemical indicators of edible plant enzymes. A suitable pH significantly promotes the proliferation of probiotics, inhibits the growth of rancid bacteria, and facilitates the accumulation of quality components during enzyme production. The pH value of the nine-steamed and nine-dried Polygonatum and black goji berry compound enzyme powder is 3.6, which may be due to the large amount of acid produced by the selected fermentation bacteria during fermentation. The polysaccharide content of the Polygonatum and goji berry compound enzyme powder is 457.31 mg / g, far exceeding the national standard of 28 mg / g. Through enzymatic hydrolysis, fermentation, and concentration drying, the polysaccharide concentration is significantly increased, thereby increasing the sweetness of the enzyme. Figure 4 ).

[0155] (2) The polyphenol content is 193.85 mg / g, which is also much higher than the national standard of 14 mg / g. As an important health-promoting ingredient and antioxidant active substance, polyphenols mainly have an astringent taste, which increases the flavor layer of the enzyme. Figure 5 ).

[0156] (3) Free amino acids are important sources of flavor substances and nutrients. Their content directly affects the taste of enzymes. The total amount of free amino acids in the enzyme products of this invention reaches 12.83 mg / g.

[0157] (4) Saponins are key functional components in Polygonatum sibiricum. Their content can directly affect the health care function of enzymes. After fermentation and concentration, the saponin content reached 51.42 mg / g.

[0158] (5) The high anthocyanin content is a key component of black goji berries, which has significant health benefits. Anthocyanins have excellent anti-aging effects, help protect eyesight, and can also resist ultraviolet radiation damage to the skin and repair damaged skin. At the same time, anthocyanins also help prevent cardiovascular diseases and many other benefits. The low pH environment formed by fermentation also helps stabilize anthocyanins. The anthocyanin content of the enzyme product in this invention reaches 96.18 mg / g.

[0159] 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 preparation method of a combination of Polygonatum sibiricum and Lycium ruthenicum ferments, characterized in that, The method comprises the following steps: (1) mixing prepared polygonatum and lycium ruthenicum according to a mass ratio of 2:1-1:1, then adding water to soak according to a ratio of 1:10 g / mL, and then homogenizing by using a tissue crushing homogenizer to obtain a homogenate; (2) adding honey to the homogenate obtained in step (1) according to an addition amount of 4-6% by mass fraction, and adding a cellulase and pectinase mixture, mixing, and then enzymatically hydrolyzing at 40-50 ℃ for 2-4 h, wherein the cellulase is added at an enzyme activity of 50-100 U / mg, and the pectinase is added at an enzyme activity of 400-800 U / mg, to obtain an enzymatic hydrolysate, which is sterilized for standby use; (3) inoculating the fermentation bacteria agent into the sterilized enzymatic hydrolysate obtained in step (2), and preparing the Rhizoma Polygonati and Lycium ruthenicum Murr. compound ferment after fermentation at a temperature of 36-38 ℃ for 48-72 h; the fermentation bacteria agent is Streptococcus thermophilus bacteria liquid, Lactobacillus plantarum bacteria liquid and Lactobacillus casei bacteria liquid; the viable bacterial count in the fermentation bacteria agent is at least 10 8 CFU / mL; the fermentation bacteria agent is obtained after mixing Streptococcus thermophilus bacteria liquid, Lactobacillus plantarum bacteria liquid and Lactobacillus casei bacteria liquid in a proportion of 1:1-2:1 by volume; and the fermentation bacteria agent is inoculated into the sterilized enzymatic hydrolysate in a proportion of 4-6% by volume. The preparation method of the streptococcus thermophilus liquid, lactobacillus plantarum liquid, and lactobacillus casei liquid is as follows: (1) inoculating streptococcus thermophilus, lactobacillus plantarum, and lactobacillus casei into MRS broth according to an inoculation amount of 1-10%, and pre-culturing at 35-37 ℃ and 150-300 r / min for 12-16 h to respectively prepare streptococcus thermophilus seed liquid, lactobacillus plantarum seed liquid, and lactobacillus casei seed liquid; (2) The S. thermophilus seed liquid, the L. plantarum seed liquid and the L. casei seed liquid obtained in step (1) are centrifuged to collect the bacterial bodies, and then diluted with normal saline to prepare S. thermophilus bacterial liquid, L. plantarum bacterial liquid and L. casei bacterial liquid with 10 8 CFU / mL of viable bacterial count. CFU / mL of viable bacterial count.

2. The method of claim 1, wherein, In step (2), the cellulase and pectinase are added according to a mass ratio of 1:

1.

3. The method of claim 1, wherein, In step (2), the honey includes but is not limited to osmanthus honey, milk vetch honey, litchi honey, and hundred flower honey.

4. The method of claim 1, wherein, The sterilization method of the enzymatic hydrolysate in step (2) is high-pressure sterilization at 121 ℃ for 10-20 min.

5. Polygonatum and lycium ruthenicum enzyme prepared by the method of any one of claims 1-4.

6. A product containing the Seuholibarbatus L. and Lycium ruthenicum Murr. complex ferment of claim 5, characterized in that, The product is food or health care products.

Images