Method for efficiently preparing moringa oleifera enzyme and application of moringa oleifera enzyme

Abstract

The invention discloses a method for efficiently preparing moringa oleifera enzyme and application of the moringa oleifera enzyme. The method comprises the following steps: A, treating fresh moringa oleifera leaves to obtain moringa oleifera leaf powder, adding water into the moringa oleifera leaf powder for size mixing, and sterilizing to obtain a moringa oleifera leaf fermentation stock solution; b, a leavening agent is added into the moringa oleifera leaf fermentation stock solution for fermentation, and fermentation liquor is obtained after fermentation is finished; and C, filtering the fermentation liquor, and carrying out spray drying on the supernate. According to the invention, the cheap moringa oleifera leaves are used as the raw material, and the moringa oleifera leaves are fermented by compounding the lactobacillus plantarum and the bifidobacterium breve, so that the effect of full fermentation in a short time is realized, the strain dosage is small, the fermentation process and the fermentation effect are simple and easy to control, and the product quality is stable.

Description

Technical Field

[0001] The present invention relates to the technical field of moringa enzyme preparation, and in particular to a method for efficiently preparing moringa enzyme and application thereof. Background Art

[0002] Sleep quality has a great impact on the body's cognitive ability, immune system, emotional stability, work efficiency and quality of life. Adequate sleep is essential for maintaining physical and mental health. In recent years, insomnia has gradually become a common social health problem. Long-term lack of sleep not only leads to cognitive problems such as amnesia, irritability, depression, inattention, fatigue, but also increases the risk of various chronic health problems, such as obesity, inflammation, diabetes and cardiovascular disease. Modern medical research has shown that insomnia is closely related to the balance regulation of excitatory and inhibitory amino acid neurotransmitters in the central nervous system of the brain. The mechanism of improving sleep is mainly related to regulating the balance of 5-hydroxytryptamine (5-HT) system, glutamate (Glu) and γ-aminobutyric acid (GABA). At present, most insomnia patients take synthetic drugs to improve their sleep, such as benzodiazepine receptor agonists, antihistamines, zolpidem and doxepin, etc. However, these drugs have certain side effects, including drug dependence, memory loss, gastrointestinal problems, and long-term use of these drugs can also lead to adverse reactions such as muscle relaxation, addiction, cognitive impairment and drug withdrawal rebound insomnia. Therefore, it is of great significance to explore new natural plant-derived bioactive drugs to improve sleep.

[0003] Moringa oleifera Lam. is a perennial tropical deciduous tree belonging to the Moringa genus of the Moringaceae family, also known as the drumstick tree and the miracle tree. The content of nutrients and functional active ingredients in Moringa is very high, and it has extremely high medicinal value and socioeconomic advantages. Studies have shown that Moringa extract can promote the body's metabolism, is conducive to the discharge of metabolic waste in the body, and relaxes the human body, thereby relieving fatigue and improving sleep, thereby helping to improve sleep. Regarding the development and application of Moringa in health foods, it is still in its infancy. Moringa seeds are mostly used as raw materials for direct consumption, or as active ingredients extracted from Moringa seeds, and the active ingredients are used as effective ingredients for health products. However, there are many types of health foods on the market, and the effects on improving sleep quality are uneven, and the cost is high and the price is expensive. Therefore, under the condition of ensuring the effect of improving sleep quality, how to develop a low-cost Moringa enzyme health food is an important direction worthy of research, and provides a commercial application path for the rational development of Moringa sleep-aiding products.

[0004] Chinese patent CN106235304A discloses a moringa enzyme and a preparation method thereof, wherein the patent comprises moringa seeds, moringa stems, and moringa leaves, and then adding water, yeast, and lactic acid bacteria to ferment for 3 months to obtain a fermented product, and then adding auxiliary materials such as licorice and wolfberry to continue fermenting for 1 month to obtain the moringa enzyme. The fermentation cycle of the patented technology exceeds 4 months, not only the production cycle is too long and the cost is high, but also the moringa enzyme obtained contains other ingredients (containing fermented products such as licorice and wolfberry), the product quality is not high, and it is difficult to achieve the purpose of low-cost development of moringa enzyme.

[0005] Chinese patent CN105614864A discloses a moringa enzyme tablet and a preparation method thereof. The patent uses moringa leaves as raw materials, and after fresh moringa leaves are powdered, water and fermentation agents are added for 17-19 hours. The fermented product is frozen to obtain freeze-dried powder, and the freeze-dried powder is granulated with water to obtain moringa enzyme tablets. The patented technology uses cheap moringa leaves as raw materials for fermentation, and the fermentation cycle is only 17-19 hours, which achieves the effect of preparing moringa enzyme at low cost and in a short cycle. However, the patented technology uses four strains of Bifidobacterium longum, Lactobacillus acidophilus, Lactobacillus delbrueckii subspecies Bulgaria and Streptococcus thermophilus as fermentation agents, and the fermentation process and fermentation effect are not well controlled, and the product quality is unstable. At the same time, the moringa enzyme tablet uses moringa enzyme as the main raw material, and the amount used reaches more than 90%, resulting in the moringa fragrance being too strong and greasy, and the taste is poor. Too much moringa enzyme is also not conducive to the low cost of the product. Summary of the invention

[0006] The purpose of the present invention is to provide a method for efficiently preparing moringa enzyme and its application in view of the above-mentioned problems. The present invention uses cheap moringa leaves as raw materials, and ferments the moringa leaves by compounding Lactobacillus plantarum and Bifidobacterium breve, which not only achieves sufficient fermentation in a short time, but also uses a small number of strains, and the fermentation process and fermentation effect are simple and easy to control, and the product quality is stable. The commercial value of moringa enzyme is achieved from three aspects: raw materials, preparation process and products, and the commercial value of moringa enzyme is developed at low cost and high efficiency, overcoming the shortcomings of existing moringa enzyme preparation and application.

[0007] The technical solution adopted by the present invention is as follows: A method for efficiently preparing Moringa enzyme comprises the following steps:

[0008] A, fresh Moringa leaves are processed to obtain Moringa leaf powder, the Moringa leaf powder is slurried with water, and then sterilized to obtain Moringa leaf fermentation stock solution;

[0009] B. After the Moringa leaf fermentation stock solution is cooled to 40±5°C, a fermentation agent is added, mixed evenly, and fermentation is performed to obtain a fermentation liquid after the fermentation is completed;

[0010] C. Filter the fermentation liquid, take the supernatant, and spray dry the supernatant to obtain the product.

[0011] In the invention, the fermentation agent is a composite strain of Lactobacillus plantarum and Bifidobacterium breve. Compared with the four strains used in patent CN105614864A, the present invention reduces the types of strains used, thereby making the fermentation process easier to control, the fermentation effect is guaranteed, and the quality of the fermented product is more stable. At the same time, the addition amount of the fermentation agent of the present invention is 0.1-0.5% (for example, it can be 0.1%, 0.12%, 0.15%, 0.2%, 0.3%, 0.5%, etc.) of the volume of the Moringa leaf fermentation stock solution, while in the patent, the inoculation amount reaches more than 5wt%. Within similar fermentation time, the present invention can fully ferment the raw materials with a smaller inoculation amount, saving the use of the fermentation agent, thereby further reducing the fermentation cost.

[0012] Further, plant lactobacillus of the present invention can specifically be GDMCC1.2685 plant lactobacillus, and Bifidobacterium breve can specifically be GDMCC1.206 Bifidobacterium breve. In the present invention, plant lactobacillus and Bifidobacterium breve are compounded not in random combination, but the present invention is obtained through multiple test screening and summary. When screening strains, the inventors have investigated strains such as plant lactobacillus, Lactobacillus salivarius, Lactobacillus yolkii, Bifidobacterium longum, and Bifidobacterium breve separately, and found that the gamma-aminobutyric acid (GABA) enriched after fermentation of plant lactobacillus and Bifidobacterium breve is the most, and the fermentation effect is the best. However, if plant lactobacillus or Bifidobacterium breve are used alone for fermentation, the fermentation period exceeds 30h, and the fermentation time is long, which is not conducive to cost control. The inventors compounded Lactobacillus plantarum and Bifidobacterium breve for fermentation tests, and found that since there was a competitive relationship between Lactobacillus plantarum and Bifidobacterium breve and they belonged to similar species, the activity of the species was improved after the competitive species was introduced, and the increase in the activity of the species prompted the fermentation process to proceed rapidly. Moreover, during the fermentation of Lactobacillus plantarum and Bifidobacterium breve, a large amount of organic acid was produced, which caused the pH value of the bacterial solution to drop rapidly. The lower the pH value, the more conducive it was to the expression of the activity of Lactobacillus plantarum and Bifidobacterium breve. The introduction of the competitive relationship and the rapid decrease in the pH value increased the activity of Lactobacillus plantarum and Bifidobacterium breve, and created the best fermentation environment in advance. In general, Lactobacillus plantarum and Bifidobacterium breve could complete the fermentation within 22 hours, and a synergistic effect was achieved.

[0013] Furthermore, the mass ratio of Lactobacillus plantarum to Bifidobacterium breve is 2:1-2, for example, 2:1, 2:1.1, 2:1.2, 2:1.5, 2:2, etc.

[0014] Furthermore, the fermentation temperature is 38-42°C and the fermentation time is 18-22h.

[0015] Further, in step A, the fresh Moringa leaves are washed, placed in a constant temperature drying oven for drying, and then crushed and sieved to obtain Moringa leaf powder, the Moringa leaf powder and water are mixed and slurried in a mass ratio of 1:5-15, and then sterilized at 90-120°C to obtain Moringa leaf fermentation stock solution.

[0016] Further, in step C, the spray drying process is: the inlet air temperature is 165-185°C, the outlet air temperature is 75-85°C, and the feed flow rate is 9-12L / min.

[0017] Furthermore, the present invention also includes a Moringa enzyme, which is prepared by the above-mentioned preparation method.

[0018] Furthermore, the present invention also includes a use of Moringa enzyme, using Moringa enzyme to prepare but not limited to powder, tablets, compressed candies, soft candies, oral liquids, capsules or beverages.

[0019] Furthermore, when Moringa enzyme is used to prepare soft candies, the mass percentages include 1.5±0.5% Moringa enzyme powder, 0.8±0.1% gellan gum, 8±1% tamarind paste, 0.3±0.1% sodium citrate, and the remainders are fructose syrup, maltose and white sugar, wherein the mass ratio of fructose syrup to maltose is 7-9:1, and white sugar is an appropriate amount.

[0020] In the above formula, it is concluded through single factor test that the addition amount of Moringa enzyme powder should not be too little or too much. Too little will not reflect the Moringa fragrance, and too much will make the Moringa too heavy, the sugar color will become darker, and the color will become more ugly. The optimal addition amount of Moringa fermentation powder is 1.5% based on the comprehensive sensory score and the actual situation. Tamarind mud mainly affects the acidity of the product. After the test, the optimal addition amount of tamarind mud is 8%. The mass ratio of fructose syrup to maltose has a great influence on the taste, appearance and shelf life of the product. When the mass ratio is 8:1, the hardness and acidity are moderate. When the amount of gellan gum is too little, the gel effect of the soft candy is poor, the soft candy is too soft and easy to stick; when the amount of gellan gum is too much, the soft candy has a good gel effect, but it is too hard and chewing is difficult; when the amount of gellan gum is 0.8%, the sensory score is the highest. The addition amount of sodium citrate affects the flavor and hardness of Moringa soft candy. It can adjust the sweet and sour taste, lower the pH of the soft candy, decompose the gelling agent, and reduce the hardness. The optimal addition amount is 0.3%.

[0021] Further, when moringa enzyme is used to prepare compressed candy, the compressed candy includes the following raw material components by mass: 40±10 parts of moringa enzyme, 15±5 parts of γ-aminobutyric acid, 25±5 parts of spinosa seed extract, ±5 parts of passion fruit extract, 3±2 parts of honey extract, 15±5 parts of corn starch, and 1.5±0.8 parts of magnesium stearate.

[0022] In summary, due to the adoption of the above technical scheme, the beneficial effects of the present invention are as follows: the present invention uses cheap Moringa leaves as raw materials, and ferments the Moringa leaves by compounding Lactobacillus plantarum and Bifidobacterium breve, which not only achieves the effect of full fermentation in a short time, but also has a small amount of strains, and the fermentation process and fermentation effect are simple and easy to control, and the product quality is stable, thereby realizing the commercial value of low-cost and efficient development of Moringa enzyme from three aspects: raw materials, preparation process and products, and overcoming the shortcomings of existing Moringa enzyme preparation and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 1 is a growth curve and pH change diagram of Lactobacillus plantarum and Bifidobacterium breve of Example 1 of the present invention;

[0024] Figure 2 The present invention is a schematic diagram of the process flow of preparing Moringa soft candy according to embodiment 3 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be described in detail below in conjunction with the accompanying drawings.

[0026] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0027] Example 1

[0028] The present embodiment provides a method for efficiently preparing Moringa enzyme, comprising the following steps:

[0029] (1) preparing materials: drying and crushing fresh Moringa leaves and sieving to obtain Moringa leaf powder;

[0030] (2) slurry preparation: slurry the moringa leaf powder and water at a ratio of 1:10 to obtain moringa leaf powder stock;

[0031] (3) Sterilization and inoculation: sterilize the Moringa leaf powder pulp, cool it to 40±5° C., transfer it into the fermentation equipment, add the composite fermentation agent and mix it to obtain the Moringa leaf powder fermentation stock solution; Moringa fermentation uses a composite strain of Lactobacillus plantarum and Bifidobacterium breve, and inoculates at an inoculation rate of 0.2%. Before inoculation, the operation is performed in a sterile room, and gloves are worn and alcohol is sprayed to avoid contamination of the strains;

[0032] (4) Fermentation: The fermentation temperature of Moringa leaves is 38-42° C., the stirring speed is 30 rpm / min, the fermentation is carried out for 18-22 hours, and the fermentation is stopped when the pH reaches 4.5;

[0033] (5) Filtration: First filter three times using gauze, then filter twice using a plate and frame filter to obtain a supernatant;

[0034] (6) Spray drying: The supernatant is spray dried with an inlet air temperature of 165-185°C, an outlet air temperature of 75-85°C, and a feed flow rate of 9-12 L / min. The collected powder is Moringa enzyme.

[0035] This example specifically investigates the effects of some strains on the fermentation and extraction of Moringa leaves, with γ-aminobutyric acid content as the evaluation index. The strains involved include: GDMCC 1.2685 Lactobacillus plantarum, GDMCC 1.986 Lactobacillus salivarius, GDMCC1.520 Bifidobacterium longum, GDMCC 1.206 Bifidobacterium breve and CICC 1.730 Lactobacillus johnsonii. The investigation is completed by the following steps to screen out the strains required for Moringa fermentation.

[0036] 1. Activation and cultivation of strains

[0037] 1. Activate the strains preserved in the laboratory, use an inoculation loop to take a small amount of bacterial liquid from the bacterial preservation tube and streak it on the MRS solid culture medium, and place the plate in an inverted state at 37°C for 48 hours. Pick a single colony and inoculate it into 5mL of MRS liquid culture medium and incubate it at 37°C for 24 hours. Then inoculate it into 10mL of MRS liquid culture medium at a 2% inoculation amount and incubate it at 37°C for 18 hours. Repeat the above steps and incubate it at 37°C for 12 hours. After completing three activations, you will get the third-generation activated bacterial suspension, which will be stored in a refrigerator at 4°C for later use.

[0038] 2. Preparation of fermentation broth

[0039] The bacterial liquid activated three times as described in 1 above was inoculated at an inoculum amount of 0.2% of the mass ratio of the original pulp of Moringa leaf powder, and fermented at 38-42°C for 48h to obtain the fermentation liquid to be tested.

[0040] 2. Determination of GABA content in fermentation broth

[0041] 1. HPLC conditions

[0042] The content of GABA in the fermentation supernatant was determined by high performance liquid chromatography and analyzed according to the national standard QBT4587-2013. The chromatographic conditions were:

[0043] Chromatographic column: C18 chromatographic column; column temperature 40℃; injection volume: 5uL;

[0044] Mobile phase A: Weigh 80g of crystalline sodium acetate and dissolve it in water to make up to 1000mL; then add 220μL of triethylamine, stir and add 5% acetic acid dropwise to adjust the pH to 7.20+0.02; finally add 5mL of tetrahydrofuran, mix and filter to obtain;

[0045] Mobile phase B: Weigh 80g of crystalline sodium acetate and dissolve it in water to make up to 1000mL; then add 2% acetic acid to adjust the pH to 7.20+0.02; then mix sodium acetate solution: acetonitrile methanol = 1:2:2 (volume ratio) and filter to obtain;

[0046] Flow rate: 1 mL / min, detection wavelength 338 nm.

[0047] 2. Sample pretreatment

[0048] Take 1 mL of the fermented sample, centrifuge it at 8000 r / min for 5 min, dilute the supernatant by a certain multiple, filter it through a 0.22 μm microporous filter membrane, and then use it for derivatization reaction.

[0049] 3. Sample derivatization

[0050] (1) Accurately weigh 2.47 g of boric acid (accurate to 00001 g), add about 80 mL of water, adjust the pH to 10.2 with sodium hydroxide, and make up to 100 mL with water;

[0051] (2) Preparation of derivatization reagent: Weigh 0.1 g of o-phthalaldehyde (OPA), dissolve it in 1 mL of acetonitrile, add 130 uL of mercaptoethanol, and dilute to 10 mL with 0.4 mol / L boric acid buffer;

[0052] (3) Pre-column derivatization: Accurately measure the specified volume of sample solution and 5.0uL of derivatization reagent, mix and react for about 2 minutes, and then inject.

[0053] 4. Drawing of standard curve

[0054] Prepare 1.0 mg / mL GABA standard solution and dilute it into 0.1, 0.5, 1, 2, 5, 10 mg / mL standard solution, and inject it after derivatization. Draw the standard curve with the peak area as the ordinate and the mass concentration of the standard solution as the abscissa, and the linear equation is Y=199012.33X-20441.28.24, R 2 =0.9994, and the linear relationship was good in the GABA mass concentration range of 0.1-10 mg / mL.

[0055] 3. Screening results

[0056] 1. Bacterial growth curve

[0057] Figure 1 The growth curves of Lactobacillus plantarum (M1) and Bifidobacterium breve (M2) and the pH changes during the fermentation process are shown in Figure 2. Figure 1From the OD600 values, it can be seen that M1 and M2 began to grow rapidly from 2 hours and entered the logarithmic growth phase. M2 grew faster, with OD600 reaching 1.5 after 12 hours and tending to grow steadily after 24 hours; M1 reached OD600 of 2.45 after 20 hours and tended to be stable after 40 hours. After M1 and M2 were cultured at 36℃ for 2 hours, the pH began to drop significantly with the growth of mycelium, and M1 tended to be stable after 48 hours, and the final pH of the fermentation broth was maintained at about 3.7; M2 tended to be stable after 32 hours, and the final pH of the fermentation broth was maintained at about 4.1.

[0058] 2. Effect of bacterial species on GABA enrichment by Moringa leaf fermentation

[0059] Table 1 Determination results of GABA enrichment by strains of Moringa leaves fermentation

[0060] Fermentation strains GABA content mg / L Lactobacillus plantarum 246.734±4.03 Lactobacillus salivarius 149.474±6.95 Lactobacillus johnsonii 134.083±4.36 Bifidobacterium longum 125.292±3.28 Bifidobacterium breve 216.653±4.83 Unfermented 103.321±3.77

[0061] As shown in Table 1, compared with the unfermented Moringa liquid, the GABA content of the fermented Moringa liquid increased to a certain extent, among which Lactobacillus plantarum and Bifidobacterium breve had the best fermentation effects, increasing by 2.4 times and 2.1 times respectively. Therefore, Lactobacillus plantarum or Bifidobacterium breve can be used as the fermentation strain. Figure 1 As a result, both fermentation cycles all exceed 30h, and fermentation time is longer, which is unfavorable for cost control.For this reason, according to the fermentation characteristics of plant lactobacillus and Bifidobacterium breve, plant lactobacillus and Bifidobacterium breve are composited as bacterial fermentation, in the case of inoculating the same bacterial species amount, obtained by test, due to the presence of interspecies competition between plant lactobacillus and Bifidobacterium breve, and belonging to close bacterial species, fermentation process can also make the pH value of bacterial liquid decline, the composite bacterial species in the fermentation process, the obvious rising of bacterial species activity, build the best fermentation environment in advance, promote the fermentation process to carry out rapidly, make the fermentation cycle just can be completed in 22h on the whole, overcome the problem that the fermentation cycle existing with plant lactobacillus and Bifidobacterium breve alone is longer, and obtain the effect of synergistic effect.Therefore, the present embodiment uses plant lactobacillus and Bifidobacterium breve composite as fermentation bacterial species, and the mass ratio of both is 2:1-2, preferably 2:1.

[0062] Example 2

[0063] 1. The present embodiment provides an application of Moringa enzyme in a health care product. The Moringa enzyme powder prepared in Example 1 is yellow-green in appearance. It is specifically applied as a Moringa enzyme compressed candy for improving sleep, and its formula is (parts by mass): 40±10 parts of Moringa enzyme, 10±5 parts of γ-aminobutyric acid, 15±5 parts of Ziziphus jujuba seed extract, 15±5 parts of passion fruit extract, 3±2 parts of honey extract, 15±5 parts of corn starch, and 1.5±0.8 parts of magnesium stearate.

[0064] Furthermore, the formula of the moringa enzyme compressed candy is preferably: 40 parts of moringa enzyme, 10 parts of γ-aminobutyric acid, 15 parts of spiny jujube seed extract, 15 parts of passion fruit extract, 3.8 parts of honey extract, 15 parts of corn starch, and 1.2 parts of magnesium stearate.

[0065] In the above, the preparation method of the Chinese jujube seed extract is: the dried Chinese jujube seeds are crushed, soaked in 10 times the amount of water for 2 hours, steamed for 2.5 hours, filtered through 100 mesh gauze, and the filtrate is reserved; the filter residue is added with water for a second steaming, boiled for 2 hours, filtered, the two filtrates are combined, and vacuum freeze-dried, the drying parameters are 8MPa, 60-70℃, the dried Chinese jujube seed concentrated paste is crushed and sieved to obtain the Chinese jujube seed extract.

[0066] Other raw and auxiliary materials, unless otherwise specified, can be purchased on the relevant market. For example, γ-aminobutyric acid was purchased from Bloomage Biotech Co., Ltd.

[0067] 2. The preparation method of the Moringa enzyme tablet for improving sleep is as follows:

[0068] S1: According to the formula ratio, weigh 4kg of Moringa enzyme, 1.5kg of Ziziphus jujuba seed extract, 1kg of γ-aminobutyric acid, 1.5kg of passion fruit extract, 0.38kg of honey extract, 1.5kg of corn starch, and 0.12kg of magnesium stearate acid, mix well, pass through an 80-mesh sieve to remove large particles and other impurities, so that the powder to be pressed is fine and the auxiliary materials absorb moisture and agglomerate to form large particles that affect tableting;

[0069] S2: using a fully automatic tablet press to compress the mixed powder obtained above into tablets to obtain tablet candies;

[0070] S3 removes residual powder on the surface of candies, and selects and removes candies with rough surfaces and irregular shapes;

[0071] S4 is packaged into sugar tablets, covered with a layer of aluminum foil, with specifications of 10 tablets / plate and 12 plates / box;

[0072] S5 performs outer packaging and then inspects the finished products. After passing the inspection, they are put into storage.

[0073] The raw materials, auxiliary materials, food additives, packaging materials and inspection rules used are in compliance with the requirements of the standard "SB / T10347-2017 Candy Tablet Candy".

[0074] 3. Production standards and determination of Moringa enzyme tablet candy

[0075] This embodiment adopts dry granulation, and optimizes the prescription by taking the disintegration degree and drying loss of the granules as indicators.

[0076] (1) Disintegration time determination method: A lifting disintegration instrument is used, the main structure of which is a lifting metal bracket and a hanging basket with a screen at the lower end, and a baffle. The lifting metal bracket moves up and down a distance of 55mm±2mm, and the reciprocating frequency is 30-32 times per minute. The specific operation is to hang the hanging basket on the metal bracket through the stainless steel shaft at the upper end, immerse it in a 1000mL beaker, and adjust the position of the hanging basket so that the screen is 25mm away from the bottom of the beaker when it descends. The beaker is filled with water at a temperature of 37℃±1℃, and the water level is adjusted so that the screen is 15mm below the water surface when the hanging basket rises. Take 6 pieces of sugar tablets to be tested, place them in the glass tubes of the above-mentioned hanging baskets, add baffles, and start the disintegration instrument for inspection. All pieces of the original powder tablets of the medicinal material should be completely disintegrated within 10 minutes. If one piece cannot be completely disintegrated, another 6 pieces should be taken for retesting, and all should meet the requirements (the compressed candy in this embodiment is measured according to the lozenge standard).

[0077] (2) Determination of loss on drying:

[0078] Crush the sample, sample diagonally using the quartering method, take about 25g, place in a clean, dry, wide-mouth bottle with a lid, mix well and set aside; accurately weigh the weighing bottle that has been dried and weighed to a constant weight, and accurately weigh about 3-5g of the sample. Place it in a vacuum drying oven, make the vacuum degree reach 0.09MPa, control the temperature at 80℃±2℃, dry for at least 4h, take out the weighing bottle, cover it and place it in a desiccator, cool it to room temperature, then take it out and weigh it, accurate to 0.0001g. Repeat the operation, heat for at least 1h and weigh it, until the mass difference obtained by two consecutive weighings does not exceed 0.002g.

[0079] The drying loss content in candy is calculated according to the following formula:

[0080] Drying loss = (total mass of weighing bottle containing sample - total mass of weighing bottle after drying) / (total mass of weighing bottle containing sample - mass of weighing bottle) × 100%

[0081] (3) Lubricant dosage screening

[0082] Taking the sticky pore degree and disintegration time as indicators, the effects of different addition amounts of magnesium stearate (0.3%, 0.6%, 0.9%, 1.2%, and 1.5%) were investigated to screen the optimal amount of lubricant.

[0083] Table 2 Effect of magnesium stearate addition

[0084] Addition amount (%) Degree of sticky hole Disintegration time (min) 0.3％ Sticky holes, difficult to form tablets — 0.6％ Sticky holes, difficult to form tablets — 0.9％ Slightly sticky holes 8.36±2.14 1.2％ Non-stick holes 12.62±0.79 1.5％ Non-stick holes 11.51±1.47

[0085] It can be seen from Table 2 that with the increase of the amount of magnesium stearate added, the degree of material sticking to the holes decreases. When the amount added is less than 0.9%, it is difficult to form tablets. When the amount added is 0.9%, the material sticks to the holes slightly, but the forming rate is low, and the difference in disintegration time is too large. When the amount added is 1.2% and 1.5%, a higher forming rate can be guaranteed, and the disintegration time meets the standard. Compared with the two, the sugar tablet with an addition of 1.2% is more stable as a whole, so the magnesium stearate addition of 1.2% is selected.

[0086] (4) Determination of quality standards of Moringa enzyme tablet candy, as shown in Table 3:

[0087] Table 3 Moringa enzyme tablet candy quality standard determination results

[0088] project in accordance with index Measured data Loss on drying / (g / 100g) SB / T 10347-2017 ≤5 2.63 Total colony count / (CFU / g) GB17399—2016 ≤1000 126 Coliform group (MPN / 100g) GB17399—2016 ≤90 Not detected

[0089] It can be seen from Table 3 that the quality of the Moringa enzyme tablet candy prepared in this embodiment meets the standard requirements. At the same time, the tablet candy prepared by the present invention meets the requirements of the Candy Product Production License Review Rules [Tablet Candy] and SB / T 10347-2017 "Candy Tablet Candy" after being tested by the Kunming Customs Technical Center (test report number: No: 01SC202101600).

[0090] Example 3

[0091] 1. This embodiment is a Moringa sleep soft candy for improving sleep, which comprises, by mass percentage, 1.5±0.5% of Moringa enzyme powder, 0.8±0.1% of gellan gum, 8±1% of tamarind paste, 0.3±0.1% of sodium citrate, and the remainder is fructose syrup and maltose, wherein the mass ratio of fructose syrup to maltose is 7-9:1.

[0092] Preferably, the raw material composition of the soft candy is: 1.5% of moringa enzyme powder, 0.8% of gellan gum, 8% of tamarind paste, 0.3% of sodium citrate, and the remainder is fructose syrup and maltose, wherein the mass ratio of fructose syrup to maltose is 8:1.

[0093] Among them, the Moringa enzyme powder is the Moringa enzyme powder prepared in Example 1, which is yellow-green.

[0094] The preparation method of tamarind mud is as follows: after peeling and core-removing clean tamarind, adding water 5 times its weight, soaking for 10 minutes, heating and boiling for extraction for 1 hour, stirring appropriately during the heating process, passing through a 350-mesh sieve after heating is completed, and concentrating to a solid content of 5wt%, namely tamarind mud.

[0095] Other raw and auxiliary materials, unless otherwise specified, can be purchased on the relevant market. For example, γ-aminobutyric acid was purchased from Bloomage Biotech Co., Ltd.

[0096] 2. The preparation method of the Moringa sleep soft candy is:

[0097] S1. Weigh each raw material according to the formula, dissolve the Moringa enzyme powder in an appropriate amount of distilled water, place it in a 45°C water bath to accelerate dissolution, and cool it to about 18°C ​​after complete dissolution to obtain a Moringa enzyme solution for standby use;

[0098] S2, add gellan gum to the Moringa enzyme solution and mix well, let stand for 10 minutes, cover the surface of the beaker with plastic wrap and place it in a water bath and heat at a constant temperature of 80°C for 25 minutes, and after the sol is fully dissolved, a colloid is obtained, which is set aside;

[0099] S3: Add fructose syrup and maltose into appropriate amount of warm water, stir until fully dissolved, then add appropriate amount of white sugar, boil on an electric stove, first over high heat and then over low heat, use a handheld refractometer to measure the sugar content, stop heating when the sugar content is appropriate, place in a water bath to keep warm, obtain sugar solution, and set aside;

[0100] S4: Mix the sugar solution and the colloid and stir evenly, add sodium citrate, let it stand and then inject it into the mold while it is hot. The process flow is as follows: Figure 2 shown.

[0101] 3. Research on the process of Moringa sleep soft candy and product quality standards

[0102] (1) Effects of raw material composition on the sensory perception of Moringa soft candy

[0103] The addition amount of Moringa fermented powder, fructose syrup, gellan gum, sodium citrate and tamarind paste were selected as the factors to be investigated. The constant factors in the single factor were set as 1.5% Moringa powder addition, 8:1 ratio of fructose syrup to maltose, 8% tamarind paste addition, 0.8% gellan gum addition and 0.3% sodium citrate addition. For the convenience of comparative analysis, the levels of each factor are shown in Table 4.

[0104] Table 4 Single factor level table

[0105]

[0106]

[0107] Response surface experiment

[0108] On the basis of the single factor experiment, taking the sensory score as the index, four factors, namely, the amount of tamarind mud, the amount of moringa fermented powder, the amount of sodium citrate and the amount of gellan gum, were selected to carry out a response surface experimental design with 4 factors and 3 levels. The specific values ​​are shown in Table 5 below:

[0109] Table 5. Response surface test factor level table

[0110]

[0111] Texture level test

[0112] The texture analyzer TPA mode was used for measurement, with a TA4 probe, a speed of 1 mm / s before and after measurement, a compression rate of 75%, a trigger rate of 5 g, a dwell time of 1 s, and each sample was measured 5 times, each time with an interval of 10 seconds, and the results were averaged. The hardness, elasticity, chewiness and recovery were mainly measured, and the measured values ​​were divided into four levels (Ⅰ, Ⅱ, Ⅲ, Ⅳ), as shown in the following table.

[0113] Table 6 Texture grade standard table

[0114]

[0115]

[0116] Sensory Rating

[0117] The scoring panel was composed of 10 experienced people. The overall sensory quality of the product was evaluated according to the standard SB / T 10021-2017 "Candy and Gel Candy". The full score is 100 points. The final score is the average of the scores of the 10 people. The proportion of each scoring indicator and the scoring standard are shown in Table 7.

[0118] Table 7 Sensory quality scoring criteria for Moringa soft candy

[0119]

[0120]

[0121] result

[0122] The amount of Moringa enzyme powder added should not be too little or too much. Too little will not reflect the Moringa aroma, and too much will make the ram too heavy, the sugar color will become darker, and the color will become more ugly. The optimal amount of Moringa fermentation powder added is 1.5% based on the sensory score and the actual situation. Tamarind mud mainly affects the acidity of the product. After the test, the optimal amount of tamarind mud is 8%. The mass ratio of fructose syrup to maltose has a great influence on the taste, appearance and shelf life of the product. When the mass ratio is 8:1, the hardness and acidity are moderate. When the amount of gellan gum is too little, the gel effect of the soft candy is poor, the soft candy is too soft and easy to stick; when the amount of gellan gum is too much, the gel effect of the soft candy is good, but it is too hard and chewing is difficult; when the amount of gellan gum is 0.8%, the sensory score is the highest. The amount of sodium citrate added affects the flavor and hardness of Moringa soft candy. It can adjust the sweet and sour taste, reduce the pH of the soft candy, decompose the gelling agent, and reduce the hardness. The optimal amount of sodium citrate added is 0.3%.

[0123] 2) Quality index testing of Moringa sleep gummies

[0124] Physical and chemical indicators and microbiological indicators

[0125] After pretreatment, the samples were tested for total reducing sugar, moisture and loss on drying in accordance with GB 28050, GB 5009.3-2016 and SB / T 10104-2017, and for lead, arsenic and mercury in accordance with GB 2762-2017. The finished soft candies were tested for Escherichia coli and total bacterial count in accordance with GB 4789.3-2016 and GB 4789.2-2016, respectively, and pathogenic bacteria were tested in accordance with SN / T 2641-2010. The test results are shown in Table 8.

[0126] Table 8 Moringa soft candy quality index test results

[0127]

[0128] It can be seen from Table 8 that the quality of the Moringa sleep soft candy prepared in this embodiment meets the standard requirements.

[0129] Effect Example 1

[0130] This experimental example is a functional test experiment of Moringa enzyme to improve sleep.

[0131] The test substance is the Moringa enzyme prepared in Example 1, which is a yellow-green powder, and the intragastric solution is prepared with normal saline. The mice are SPF Kunming mice, and the injection dose of sodium pentobarbital for mice is determined to be 45 mg / kg·BW by preliminary experiments. Each group of mice was given the corresponding test substance, and was continuously intragastrically gavaged for 14 days. They were fasted but not watered 12 hours before the last intragastric gavage. The mice were raised under controlled environmental conditions, with constant temperature (24±1°C), relative humidity (50±10%), 12 / 12 hour light and dark cycle, and free drinking water and food intake. Before the experiment, the mice were weighed and then randomly divided into 4 groups, namely, a blank control group (95% normal saline), a low-dose group of Moringa enzyme (50 mg / kg·BW), a high-dose group (200 mg / kg·BW) and a positive group (DZP2mg / kg·BW), and an intragastric volume of 0.1mL / 10g·BW. Before the experiment, the mice were fed for 7 days, and then gavaged once a day for 14 consecutive days. The mice were weighed before gavage. Four experiments were observed and recorded, including direct sleep experiment, prolonged pentobarbital sodium sleep time experiment, pentobarbital sodium subthreshold dose hypnosis experiment, and pentobarbital sodium sleep latency experiment, and conclusions were drawn.

[0132] Experimental Results

[0133] The mice were weighed at the beginning and end of the experiment, and the weight changes of each group of mice before and after the experiment were observed. The experimental results showed that P>0.05 for the low-dose Moringa leaf enzyme group (50 mg / kg·BW), high-dose group (200 mg / kg·BW), blank control group (95% saline), and diazepam positive group (2 mg / kg·BW), and the initial and final weight changes were consistent with normal growth changes, indicating that Moringa enzyme had no significant adverse effects on mouse weight.

[0134] Furthermore, when exploring the direct effect of moringa enzyme on the sleep of mice, the experimental results were as follows: in the low-dose and high-dose groups of moringa enzyme and the blank control group, the number of animals that fell asleep was 0, and the sleep time was 0 minutes, indicating that pure moringa leaf enzyme had no significant direct sleep effect on mice.

[0135] Furthermore, when exploring the effect of moringa enzyme on the sleep time of mice induced by sodium pentobarbital, the experimental results were as follows: compared with the blank control group (23.97±2.86) min, the sleep time of the low-dose moringa leaf enzyme group was (28.64±3.99) min, and the sleep time of the high-dose group was (36.55±4.22) min. Both high and low doses could slightly prolong the sleep of mice. Compared with the normal control group, the sleep time of the oral diazepam group (diazepam) increased significantly (41.74±5.43) min, which was significantly different from the normal group. The diazepam group had a longer calming and sleeping period than the high-dose moringa leaf enzyme group. This shows that a certain dose of moringa leaf enzyme has a synergistic effect with sodium pentobarbital and can prolong the sleep time of mice.

[0136] Furthermore, when exploring the effect of Moringa enzyme on the subthreshold hypnotic effect of sodium pentobarbital, the experimental results were: the incidence of sleep in the low-dose group was 25%, the incidence of sleep in the high-dose group was 62.5%, and the control group was 12.5%. The high-dose effect was better than the low-dose effect, and the diazepam effect was better than the high-dose effect.

[0137] Furthermore, when exploring the effect of moringa enzyme on the sleep latency of mice induced by sodium pentobarbital, the experimental results were as follows: compared with the blank control group, the sleep latency of the low-dose moringa enzyme group was slightly reduced, but there was no significant difference; the sleep latency of mice in the diazepam positive control group (3.70±0.44) min was significantly lower than that of the blank control group (5.85±0.53) min, and the sleep latency of mice in the low- and high-dose moringa enzyme groups shortened with increasing doses, which were (5.20±0.83) and (3.93±0.34) min, respectively; the low- and high-dose moringa enzyme groups were significantly different from the blank control group (P<0.05). This shows that moringa enzyme and sodium pentobarbital have a synergistic effect, and the synergistic effect of 50 and 200 mg / kg doses of moringa leaf enzyme and a certain dose of sodium pentobarbital can shorten the sleep latency of mice.

[0138] Furthermore, after each group of test substances were gavaged to mice continuously for 21 days, experimental observations revealed that within 60 minutes of the last gavage, none of the mice in each group experienced direct sleep, which means that Moringa enzyme and GABA had no direct hypnotic effect on mice.

[0139] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for efficiently preparing Moringa enzyme, characterized in that, The steps include: A, fresh Moringa leaves are processed to obtain Moringa leaf powder, the Moringa leaf powder is slurried with water, and then sterilized to obtain Moringa leaf fermentation stock solution; B. After the Moringa leaf fermentation stock solution is cooled to 40±5°C, a fermentation agent is added, mixed evenly, and fermentation is performed to obtain a fermentation liquid after the fermentation is completed; C. Filter the fermentation liquid, take the supernatant, and spray dry the supernatant to obtain the product.

2. The preparation method according to claim 1, characterized in that The fermentation agent is a composite bacterial species mixed with Lactobacillus plantarum and Bifidobacterium breve, and the mass ratio of Lactobacillus plantarum to Bifidobacterium breve is 2:1-2.

3. The method according to claim 2, characterized in that The amount of the fermentation agent added is 0.1-0.5% of the volume of the Moringa leaf fermentation stock solution.

4. The method according to claim 3, characterized in that The fermentation temperature is 38-42°C, and the fermentation time is 18-22h.

5. The method according to any one of claims 1 to 4, characterized in that: In step A, fresh Moringa leaves are washed, placed in a constant temperature drying oven for drying, and then crushed and sieved to obtain Moringa leaf powder, the Moringa leaf powder and water are mixed and slurried in a mass ratio of 1:5-15, and then sterilized at 90-120° C. to obtain Moringa leaf fermentation stock solution.

6. The method according to claim 5, characterized in that In step C, the spray drying process is: the inlet air temperature is 165-185°C, the outlet air temperature is 75-85°C, and the feed flow rate is 9-12 L / min.

7. A Moringa enzyme, characterized in that, The Moringa enzyme is prepared by any of the methods described in claims 1-6 above.

8. A use of Moringa enzyme, characterized in that, Use Moringa enzyme to prepare powders, tablets, compressed candies, gummies, oral liquids, capsules or beverages.

9. The use according to claim 8, characterized in that When using Moringa enzyme to prepare soft candies, the mass percentages include 1.5±0.5% Moringa enzyme powder, 0.8±0.1% gellan gum, 8±1% tamarind paste, 0.3±0.1% sodium citrate, and the remainder are fructose syrup, maltose and white sugar, wherein the mass ratio of fructose syrup to maltose is 7-9:

1.

10. The use according to claim 8, characterized in that Moringa enzyme is used to prepare compressed candies, which include the following raw material components by mass: 40±10 parts of moringa enzyme, 15±5 parts of γ-aminobutyric acid, 25±5 parts of spinosa jujube seed extract, ±5 parts of passion fruit extract, 3±2 parts of honey extract, 15±5 parts of corn starch, and 1.5±0.8 parts of magnesium stearate.

Images