Moringa oleifera branch polyphenol extract as well as extraction method and application thereof

Abstract

The invention discloses a moringa oleifera branch polyphenol extract as well as an extraction method and application thereof, and belongs to the technical field of plant extraction. The preparation method comprises the following steps: firstly, mixing proline and lactic acid to obtain an extraction solvent, then uniformly mixing the extraction solvent with moringa oleifera branches, carrying out ultrasonic extraction, and carrying out solid-liquid separation to obtain a liquid which is the polyphenol extract. The method for extracting the moringa oleifera branch polyphenol by using the compound solvent formed by the proline and the lactic acid is green and environment-friendly, the extracted product is rich in polyphenol, meanwhile, the proline and the lactic acid are contained as feed additives, protein synthesis can be promoted, the growth performance can be improved, sustainable and green breeding production is achieved, and the moringa oleifera branch polyphenol is suitable for large-scale production. And animal health and breeding benefits are improved.

Description

Technical Field

[0001] The invention belongs to the technical field of plant extraction, and particularly relates to a Moringa branch polyphenol extract and an extraction method and application thereof. Background Art

[0002] Moringa branches are forestry waste generated during the harvesting of Moringa leaves. Although their polyphenol content is not as high as that of Moringa leaves, the biomass of Moringa branches is about 2-4 times that of leaves. As feed additives, polyphenols can promote animal health, enhance immunity, improve digestion and metabolic functions, and improve production performance in various ways. As natural antioxidants, they can effectively reduce oxidative stress and inflammatory responses and enhance the disease resistance of animals. Therefore, it is necessary to develop a method for effectively extracting polyphenols from Moringa branches.

[0003] At present, polyphenols in Moringa are usually extracted by ethanol. For example, a Chinese patent discloses a method for extracting Moringa polyphenols by low-temperature ultrasound, and its publication number is CN 107307416A. The crude extraction of Moringa leaf polyphenols: pick Moringa leaves, dry them at 80 ° C to a moisture content of 6%, grind them into powder with a magnetic stirrer, pass through an 80-mesh sieve, obtain Moringa leaf powder, and dry and store at low temperature for later use. Weigh 1 g of Moringa powder correctly, add 20 mL of ethanol at a solid-liquid ratio of 1:20 (m / v), let it stand for 30 min, and then perform ultrasound. After the ultrasound is completed, filter it with filter paper, and store the filtrate with a number; the purification method is: dissolve the Moringa leaf polyphenol crude extract solution with 25% ethanol, pass it through a D101 macroporous resin column, and then elute it with 75% ethanol. Collect the eluate at 4 mL / tube, place it in a rotary evaporator (speed 6, water temperature 37 ° C, rotary evaporation 40 min) to evaporate, and obtain the purified Moringa polyphenols. However, this ethanol extraction method has a low extraction rate, complicated steps, causes environmental pollution, and requires purification after extraction.

[0004] Therefore, there is an urgent need to develop an environmentally friendly method for extracting polyphenols that can reduce the cost of subsequent separation and purification so that the extracted products have better comprehensive utilization value. Summary of the invention

[0005] The first technical problem to be solved by the present invention is to provide a method for extracting a polyphenol extract of Moringa oleifera branches, which is green and environmentally friendly, and the extracted product is rich in polyphenols; the second technical problem to be solved by the present invention is to provide the application of the polyphenol extract of Moringa oleifera branches in a composite feed additive.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A method for extracting a polyphenol extract from a Moringa branch comprises the following steps: mixing proline and lactic acid to obtain an extraction solvent, then uniformly mixing the extraction solvent with the Moringa branch for ultrasonic extraction, and separating the solid from the liquid to obtain a polyphenol extract.

[0008] Furthermore, the molar ratio of proline to lactic acid is 1:0.5-10.

[0009] Furthermore, the extraction solvent obtained by mixing proline and lactic acid is heated in an oil bath at 80° C. and 120 rpm until a transparent liquid is formed.

[0010] Furthermore, the Moringa branches are crushed and then sieved, and the sieve mesh number is 40-80 meshes.

[0011] Furthermore, the mass ratio of the extraction solvent to the Moringa branches is 10-80:1.

[0012] Furthermore, the ultrasonic time is 20 to 60 min.

[0013] Furthermore, the ultrasonic temperature is 40-80 °C.

[0014] Furthermore, the mesh size of the filter cloth for solid-liquid separation is 300 meshes.

[0015] Furthermore, the method for extracting the Moringa branch polyphenol extract prepares the Moringa branch polyphenol extract.

[0016] Furthermore, the Moringa branch polyphenol extract is used in a composite feed additive.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] 1) The present invention uses a solvent composed of natural small molecules existing in the plant matrix as a medium and couples it with ultrasonic extraction, which can not only greatly improve the extraction effect of polyphenols in Moringa branches, but also is environmentally friendly because the raw materials are all green, non-toxic and naturally degradable.

[0019] 2) The polyphenol extract prepared by the present invention can greatly reduce the cost of separation and purification in the later stage of polyphenol extraction on the one hand, and on the other hand, the polyphenols in the extract account for 17.64 mg GAE / g DW, and the extract also contains proline and lactic acid components, which can be used as feed additives to promote protein synthesis and improve growth performance, realize sustainable and green breeding production, and improve animal health and breeding benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the effect of different ultrasonic times on the polyphenol content of the extract of the present invention;

[0021] Figure 2 This is a graph showing the effect of different ultrasonic temperatures on the polyphenol content of the extract of the present invention;

[0022] Figure 3 This is a graph showing the effect of different molar ratios of proline to lactic acid on the polyphenol content of the extract of the present invention;

[0023] Figure 4 This is a graph showing the effect of different mass ratios of Pro-LA to Moringa branches on the polyphenol content of the extract. DETAILED DESCRIPTION

[0024] The present invention is further illustrated below in conjunction with specific examples. The examples are implemented based on the technical solutions of the present invention. It should be understood that these examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0025] The method for determining the polyphenol content in the following examples is as follows: the total phenol content is determined using the Folin-Ciocalteu colorimetric method, 10 μL of the extract supernatant diluted 100 times is mixed with 50 μL of 0.1 M Folin-Ciocalteu reagent in a 96-well microplate, and then incubated at 30°C for 5 min. Afterwards, 50 μL of 10% Na2CO3 and 90 μL of distilled water are added to each well and mixed, and incubated at 30°C for another 10 min. The absorbance is recorded at λ=760 nm. A control tube is set up for each sample. The control tube uses an equal volume of distilled water instead of the Folin-Ciocalteu reagent to perform the same operation. The polyphenol content determination result will also subtract the measured value of the control tube to remove the influence of the solvent and the sample color. Different concentrations of gallic acid (2.5-156.25 μg / mL, R 2 =0.9998) were expressed as milligrams of gallic acid equivalents (GAE) per gram of dry weight of the sample (mg GAE / g DW).

[0026] Example 1

[0027] A method for extracting Moringa branch polyphenol extract, the specific steps are as follows:

[0028] (1) Crush the Moringa branches into small particles of 40-80 mesh.

[0029] (2) Proline and lactic acid were mixed in a molar ratio of 1:1, and 30% of water was added to the mixture. The mixture was heated in an oil bath at 80 °C and 120 rpm until a transparent liquid was formed, thereby obtaining the extraction solvent Pro-LA.

[0030] (3) Mix 10 g Pro-LA and 0.5 g absolutely dry Moringa branch powder in a 30 mL screw-capped glass bottle, place the mixture in an ultrasonic cleaner SB-5200DTD, extract at 360 W and 40 °C for 45 min, and perform preliminary solid-liquid separation on the extract using a double-layer 300-mesh filter cloth. The resulting liquid portion is the composite feed additive. The polyphenol content in the composite feed additive was measured to be 0.8820 mg GAE / mL.

[0031] (4) The liquid portion of step (3) was centrifuged at 10,000 rpm for 10 min to obtain the supernatant for subsequent polyphenol content determination. The polyphenol content of the supernatant finally extracted and measured was 17.64 mg GAE / g DW, the proline content was 0.43 g / g compound feed additive, and the lactic acid content was 0.34 g / g compound feed additive.

[0032] Example 2

[0033] A method for extracting Moringa branch polyphenol extract, the specific steps are as follows:

[0034] The difference from Example 1 is that the ultrasonic time in step (3) is 15 min, 30 min, 60 min, 75 min, and 90 min, and Moringa branch polyphenols are extracted. The analysis of the contents at different ultrasonic times is shown in Tables 1 and Figure 1 .

[0035] Table 1 Analysis of different ultrasonic times on various contents

[0036]

[0037] Table 1 and Figure 1 The effect of different ultrasonic time on the contents of polyphenol extracts from Moringa oleifera branches can be seen. As the extraction time increases, the polyphenol content of the extract increases first and then decreases. This is because at the beginning, with the influence of ultrasonic cavitation and the extraction of the solvent, the polyphenols gradually dissolve, but as the ultrasonic time continues to increase, the easily degradable part of the polyphenols gradually degrades. Therefore, the best ultrasonic time is 45 min.

[0038] Example 3

[0039] A method for extracting Moringa branch polyphenol extract, the specific steps are as follows:

[0040] The difference from Example 1 is that the ultrasonic temperature in step (3) is 50°C, 60°C, 70°C, and 80°C to extract Moringa branch polyphenols. The analysis of the effects of different ultrasonic temperatures on the contents is shown in Tables 2 and Figure 2 .

[0041] Table 2 Analysis of different ultrasonic temperatures on various contents

[0042]

[0043] Table 2 and Figure 2 The effects of different ultrasonic temperatures on the contents of polyphenol extracts from Moringa oleifera branches are shown in Figure 2. When the temperature is too high, the polyphenol content begins to decrease. This is because most polyphenols begin to degrade when the temperature exceeds 50 °C, so the extraction temperature should not be too high. In summary, the extraction temperature was selected to be 40 °C.

[0044] Example 4

[0045] A method for extracting Moringa branch polyphenol extract, the specific steps are as follows:

[0046] The difference from Example 1 is that the molar ratio of proline to lactic acid in step (2) is 1:0.5, 1:2, 1:4, 1:8, and 1:20, and Moringa branch polyphenols are extracted. The analysis of the effects of different molar ratios of proline to lactic acid on the contents is shown in Tables 3 and Figure 3 .

[0047] Table 3 Analysis of the effects of different proline to lactic acid molar ratios on the contents

[0048]

[0049] Table 3 and Figure 3 The influence of different molar ratios of proline and lactic acid on various data of polyphenol extracts can be seen. It can be seen that with the increase of the molar ratio, the content of polyphenol extracts shows a trend of first increasing and then decreasing. This is because the excellent extraction ability of the configured extraction solvent is due to its wide polarity range and strong internal hydrogen bond network. The continuous increase in the molar ratio destroys its hydrogen bond network, making it impossible to loosen the structure of Moringa branches and release more polyphenols. As a composite feed additive, its palatability and ratio should also be appropriate. The recommended feed addition amount of proline and lactic acid is usually between 0.1% and 0.5%. So in summary, the molar ratio is selected as 1:1.

[0050] Example 5

[0051] A method for extracting Moringa branch polyphenol extract, the specific steps are as follows:

[0052] The difference from Example 1 is that the mass ratio of Pro-LA to Moringa branches in step (3) is 10:1, 40:1, 60:1, and 80:1, and Moringa branch polyphenols are extracted. The analysis of the contents of different mass ratios of Pro-LA to Moringa branches is shown in Tables 4 and Figure 4 .

[0053] Table 4 Analysis of the contents of different Pro-LA and Moringa branches by mass ratio

[0054]

[0055] Table 4 and Figure 4 This is the effect of different Pro-LA and Moringa branch mass ratios on various data of polyphenol extracts. With the increase of liquid-to-solid ratio, the total polyphenol content in the extract increases significantly in the process of 10:1-20:1. With the further increase of liquid-to-solid ratio, it can be observed that the increase of liquid-to-solid ratio shows a slow trend, and the increase is getting smaller and smaller. In addition, with the increase of liquid-to-solid ratio, the polyphenol content per milliliter of polyphenol extract decreases rapidly. In summary, the case of liquid-to-solid ratio of 20:1 is selected as the best choice.

[0056] Comparison document 1

[0057] The ethanol reflux method was used to determine the content of polyphenols in Moringa. 10 g of 40-80 mesh Moringa branch powder was weighed, wrapped with filter paper and placed in a reflux device with an ethanol concentration of 95%. The temperature of the heating stirring table was set to 80 °C and refluxed for 2 h. The liquid was collected and its polyphenol content was measured to be 6.12 mg GAE / g DW.

[0058] Comparison document 2

[0059] 10 g of 85% lactic acid and 0.5 g of absolutely dry Moringa branch powder were mixed evenly in a 30 mL screw-mouth glass bottle, placed in an ultrasonic cleaner SB-5200DTD, and extracted at 360 W and 40 °C for 45 min. The liquid portion was taken and its polyphenol content was measured to be 5.95 mg GAE / g DW, and the lactic acid content was 0.85 g / g.

[0060] Comparison document 3

[0061] 10 g of distilled water and 0.5 g of absolutely dry Moringa branch powder were mixed evenly in a 30 mL screw-mouth glass bottle, placed in an ultrasonic cleaner SB-5200DTD, and extracted at 360 W and 40 °C for 45 min. The liquid portion was taken and its polyphenol content was measured to be 6.48 mg GAE / g DW.

[0062] Comparison document 4

[0063] 10 g of 60% methanol and 0.5 g of absolutely dry Moringa branch powder were mixed evenly in a 30 mL screw-mouth glass bottle, placed in an ultrasonic cleaner SB-5200DTD, and extracted at 360 W and 40 °C for 45 min. The liquid part was taken and its polyphenol content was measured to be 5.92 mg GAE / g DW, and the methanol content was 0.6 g / g.

[0064] Comparative Example 5

[0065] 10 g of 60% ethanol and 0.5 g of absolutely dry Moringa branch powder were mixed evenly in a 30 mL screw-mouth glass bottle, placed in an ultrasonic cleaner SB-5200DTD, and extracted at 360 W and 40 °C for 45 min. The liquid part was taken and its polyphenol content was measured to be 5.82 mg GAE / g DW, and the ethanol content was 0.6 g / g.

[0066] Example 6

[0067] A total of 30 one-day-old white-feathered broilers were purchased from a local producer, and all the broilers used in the experiment were male broilers. The feeding method was flat-raising, and the fence specifications were 160 cm×60 cm×100 cm. The broiler house was well ventilated and well lit, and the relative humidity was maintained at 55%~65%. During the entire experiment, sufficient drinking water and daily diet were provided for broilers to drink and eat. The broilers were vaccinated on the first day and then pre-fed for 7 days to adapt to the growth environment. During this period, all broilers were fed a basic diet. The broilers were weighed on the 7th day, and then the 30 white-feathered broilers were randomly divided into two treatment groups. From the 8th day until the end of the experiment, the control group was fed with a basic diet, and the other group was fed with a basic diet supplemented with 1.5% by mass.

[0068] At the end of the feeding period (day 49), 4 broilers were randomly selected from each group for sampling. The measured indicators were body weight (BW, g), average daily gain (ADG, g / d), and feed-to-meat ratio (FCR).

[0069] Calculation formula: ADG (kg / d) = (weight at the end of the test - weight at the beginning of the test) / number of feeding days; ADFI (kg / d) = total feed intake / number of feeding days.

[0070] The results showed that the final body weight of the group using the compound feed additive was 3.932±0.051 kg, the average daily weight gain was 0.090±0.003 kg, and the feed-to-meat ratio was 1.564±0.044 kg / d; the final body weight of the control group was 3.923±0.023 kg, the average daily weight gain was 0.089±0.003 kg, and the feed-to-meat ratio was 1.602±0.020 kg / d.

[0071] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting Moringa branch polyphenol extract, characterized in that, Proline and lactic acid are mixed to obtain an extraction solvent, and then the extraction solvent is mixed with Moringa branches for ultrasonic extraction. After solid-liquid separation, the obtained liquid is a polyphenol extract.

2. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The molar ratio of proline to lactic acid is 1:0.5-10.

3. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The extraction solvent obtained by mixing proline and lactic acid is heated in an oil bath at 80° C. and 120 rpm until a transparent liquid is formed.

4. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The Moringa branches are crushed and then sieved, and the sieve mesh number is 40-80 meshes.

5. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The mass ratio of the extraction solvent to the Moringa branches is 10-80:

1.

6. The method for extracting Moringa branch polyphenol extract according to claim 1, characterized in that: The ultrasonic time is 20 to 60 min.

7. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The ultrasonic temperature is 40-80°C.

8. The method for extracting Moringa branch polyphenol extract according to claim 1, wherein: The mesh number of the filter cloth for solid-liquid separation is 300 meshes.

9. The Moringa branch polyphenol extract is prepared according to the extraction method of any one of claims 1 to 8.

10. Use of the Moringa branch polyphenol extract according to claim 9 in a composite feed additive.

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