Method for extracting fresh moringa oleifera leaf protein by adding lactobacillus plantarum for fermentation

Abstract

The invention provides a method for extracting fresh moringa oleifera leaf protein by adding lactobacillus plantarum for fermentation, which comprises the following steps: cutting fresh moringa oleifera leaves into sections as raw materials to extract and process leaf protein, and sequentially carrying out the procedures of pretreatment, pulping, leaf pulp separation, fermentation, centrifugal separation, freeze drying and the like to realize complete extraction of the moringa oleifera leaf protein. The processing process is safe, simple and convenient, byproducts can be recycled for the second time, no waste is generated, and the method is environment-friendly and pollution-free; the leaf protein concentrate extraction rate can reach 59.25%, the protein content can reach 54.42%, and the leaf protein feed is a high-quality protein feed.

Description

Technical Field

[0001] The invention belongs to the technical field of deep processing and high value of moringa leaves, and specifically relates to a method for extracting fresh moringa leaf protein by adding plant lactobacillus and fermenting. Background Art

[0002] Plant leaf protein is a concentrated protein, which is a protein product extracted from the fresh stems and leaves of green plants. It has high nutritional value, a protein content of 50%-80%, and is rich in amino acids required by the human body, among which the content of eight essential amino acids is relatively high. As one of the world's most renewable protein resources, plant stem and leaf protein extracts and processed products are widely used in food, cosmetics, feed, pesticides, medicine and plant growth regulators. Leaf protein can also be used as a raw material for further separation to produce active substances with different functions, such as pure protein, carotene, chlorophyll and vitamins, to achieve the effects of strengthening the body, preventing diseases and anti-aging. According to different precipitation processes, the crude extracted leaf protein is divided into albumin and green protein. Among them, the main component of albumin is soluble protein, which is used as a human food additive; the main component of green protein is insoluble protein, which is generally used as livestock feed.

[0003] Moringa is a tropical plant with unique economic value. It is native to India and is widely planted in tropical and subtropical regions of Asia, Africa, and Central America. It grows fast, has a large biomass, and has the characteristics of rapid growth and high yield. Moringa, also known as the vegetable tree, can be eaten as vegetables with its fresh leaves, twigs, and fruit pods. It is rich in active ingredients and can not only provide abundant protein, vitamins, and minerals, but also play the role of antioxidant, anti-inflammatory, anti-diabetic, lipid-lowering, anti-cancer, and antibacterial functions, promoting human metabolism, strengthening physical fitness, and improving immunity.

[0004] As the main edible part of Moringa, leaves are rich in nutrients such as protein, vitamins, minerals, and low in fat and anti-nutritional factors. The amino acid composition of leaf protein is balanced, with a total of 24.27%, including 9 essential amino acids, with higher contents of glutamic acid, aspartic acid, and threonine. Studies have shown that the protein and amino acid content of extracted Moringa leaves is significantly increased compared with unextracted leaves, and its chlorophyll extraction provides an important way to develop protein feed resources for monogastric animals in short supply.

[0005] The extraction method of Moringa leaf protein is mainly based on the conventional plant protein extraction method, which mainly includes the following three steps: destroying tissue by mechanical treatment, precipitation, and concentrating protein by one or more heating and / or pH treatment, mostly using acid-base heating method, salting-out method, ultrafiltration method, etc. Patent CN106987611A provides a method for preparing Moringa leaf protein polypeptide by microwave-assisted and membrane filtration, and uses microwave-assisted biological enzymolysis to prepare Moringa leaf protein polypeptide. The reaction conditions are mild, easy to operate, low in energy consumption, short in enzymolysis time, no solvent is required, the product is safe and of good quality, and it improves the palatability of protein and improves digestibility. Patent CN111248341A provides a Moringa leaf protein powder feed for improving fish quality and a preparation method thereof, which increases the crude protein content and trace element content of fish, increases the nutritional value of fish, increases the umami taste of fish, and improves the taste of fish.

[0006] However, both the acid-heat method and the alkali-heat method will cause a certain degree of environmental pollution, and will cause changes in the smell and color of the final leaf protein product, and there are energy consumption problems; the salting-out method needs to be used in conjunction with other extraction methods to increase the extraction rate, and the leaf protein quality is poor; the organic solvent method is difficult to recover the organic solvent during the extraction process, and the residual problem is serious. Summary of the invention

[0007] The invention aims to provide a method for extracting fresh Moringa leaf protein by adding Lactobacillus plantarum to ferment, which improves the extraction efficiency and quality of leaf protein through pretreatment, pulping, leaf pulp separation, adding Lactobacillus plantarum to ferment, separation and freeze drying and other processes.

[0008] In order to achieve the above object, the present invention provides the following technical solutions:

[0009] A method for extracting fresh Moringa leaf protein by adding Lactobacillus plantarum and fermenting, comprising the following steps:

[0010] S1: Pretreatment: clean fresh Moringa leaves with water, remove impurities, cut into sections for later use;

[0011] S2: beating: put the fresh Moringa leaves obtained in step S1 into a beating machine, add water and beat to obtain freshly squeezed leaf pulp;

[0012] S3: Leaf pulp separation: freshly squeezed leaf pulp is filtered to separate leaf residue and green juice; the cleaning liquid from the pulping machine and the leaf residue are mixed and filtered to collect all the leaf residue and green juice;

[0013] S4: fermentation: adding Lactobacillus plantarum to the green juice obtained in step S3, mixing evenly, placing in a constant temperature incubator for anaerobic fermentation to obtain a fermentation liquid;

[0014] S5: centrifugal separation: placing the fermentation liquid obtained in step S4 in a centrifuge for centrifugal separation to obtain leaf protein precipitate;

[0015] S6: Freeze drying: The leaf protein precipitate obtained in step S5 is placed in a freeze dryer for freeze drying to obtain a finished leaf protein product.

[0016] Furthermore, step S1 is specifically as follows: washing the picked fresh Moringa leaves with clean water to remove impurities such as rotten leaves, mud and sand; cutting the washed fresh Moringa leaves into small segments of 1-2 cm for later use.

[0017] Furthermore, in step S2, the cut fresh Moringa leaves are placed in a pulper, and water is added at a solid-liquid ratio of 1:13 for pulping for 2-3 minutes to obtain freshly squeezed leaf pulp.

[0018] Furthermore, in step S3, a 300-mesh nylon mesh filter bag is used to filter the freshly squeezed leaf pulp to separate the leaf residue and the green juice; the pulper is cleaned with deionized water, the obtained cleaning liquid is combined with the leaf residue, and filtered again with a 300-mesh nylon mesh filter bag to collect all the leaf residue and green juice.

[0019] Furthermore, in step S4, 4×10 6 cells / mL of Lactobacillus plantarum was added to the leaf juice and stirred at room temperature for 3 minutes.

[0020] Furthermore, step S4 is placed in a constant temperature incubator at 30° C. for anaerobic fermentation for 21 hours. Through fermentation, Lactobacillus plantarum promotes the fermentation of Moringa green juice, causing it to produce acid during the fermentation process, lowering the pH to reach the isoelectric point, and promoting protein precipitation.

[0021] Furthermore, in step S5, the fermentation liquid is placed in a centrifuge and centrifuged at a rate of 5000 r / min for 15 minutes to obtain leaf protein precipitation.

[0022] Furthermore, in step S5, the leaf protein precipitate is placed in a -60°C freeze dryer for 12 hours to obtain a leaf protein product.

[0023] Compared with the prior art, the advantages and beneficial effects of the present invention are:

[0024] 1. The present invention uses fresh Moringa leaf segments as raw materials to extract and process leaf protein. Compared with the research using Moringa leaf dry powder as experimental material, it saves raw material processing technology and significantly reduces actual production costs.

[0025] 2. In the deep processing of Moringa oleifera, the present invention sequentially undergoes processes such as pretreatment, pulping, leaf pulp separation, adding Lactobacillus plantarum fermentation, separation freeze drying, etc., with complete extraction and a safe and simple processing process without chemical pollution, pure natural and no waste generated, and all by-products can be recycled for secondary use.

[0026] 3. In order to better promote the extraction quality and efficiency of leaf protein, the present invention introduces plant lactobacillus to promote the fermentation of Moringa green juice, so that it produces acid during the fermentation process, reduces the pH to the isoelectric point, and promotes protein precipitation. The prepared leaf protein concentrate has a high extraction rate and high protein content, and can serve as a high-quality protein feed. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Response surface diagram and contour diagram of the effect of leaf protein extraction rate in Example 1. DETAILED DESCRIPTION

[0028] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0029] Example 1

[0030] The present embodiment provides a method for extracting protein from fresh Moringa leaves by adding Lactobacillus plantarum and fermenting, comprising the following steps:

[0031] S1: Pretreatment: Wash the freshly picked Moringa leaves with clean water to remove impurities such as rotten leaves and sand; cut the washed Moringa leaves into small pieces of 1-2 cm for later use;

[0032] S2: Pulping: Put the cut fresh Moringa leaves into a pulper, add water at a solid-liquid ratio of 1:10 and pulp for 2 minutes to obtain freshly squeezed leaf pulp;

[0033] S3: Leaf pulp separation: Use a 300-mesh nylon mesh filter bag to filter the freshly squeezed leaf pulp to separate the leaf residue and green juice; use deionized water to clean the pulper, combine the obtained cleaning liquid with the leaf residue, and filter again with a 300-mesh nylon mesh filter bag to collect all the leaf residue and green juice;

[0034] S4: Fermentation: Add 3.5×10 Lactobacillus plantarum to the green juice obtained in step S3 6 / mL, stir at room temperature for 3 minutes to mix evenly, and place in a 30℃ constant temperature incubator for anaerobic fermentation to obtain the fermentation liquid;

[0035] S5: centrifugal separation: the fermentation liquid obtained in step S4 is placed in a centrifuge and centrifuged at a rate of 4500 r / min for 12 min to obtain leaf protein precipitate;

[0036] S6: Freeze drying: The leaf protein precipitate obtained in step S5 is placed in a -60°C freeze dryer and freeze-dried for 12 hours to obtain a finished leaf protein product.

[0037] Example 2

[0038] The present embodiment provides a method for extracting protein from fresh Moringa leaves by adding Lactobacillus plantarum and fermenting, comprising the following steps:

[0039] S1: Pretreatment: Wash the freshly picked Moringa leaves with clean water to remove impurities such as rotten leaves and sand; cut the washed Moringa leaves into small pieces of 1-2 cm for later use;

[0040] S2: Pulping: Put the cut fresh Moringa leaves into a pulper, add water at a solid-liquid ratio of 1:15 and pulp for 3 minutes to obtain freshly squeezed leaf pulp;

[0041] S3: Leaf pulp separation: Use a 300-mesh nylon mesh filter bag to filter the freshly squeezed leaf pulp to separate the leaf residue and green juice; use deionized water to clean the pulper, combine the obtained cleaning liquid with the leaf residue, and filter again with a 300-mesh nylon mesh filter bag to collect all the leaf residue and green juice;

[0042] S4: Fermentation: Add 5×10 Lactobacillus plantarum to the green juice obtained in step S3 6 / mL, stir at room temperature for 3 minutes to mix evenly, and place in a 35℃ constant temperature incubator for anaerobic fermentation to obtain the fermentation liquid;

[0043] S5: centrifugal separation: the fermentation liquid obtained in step S4 is placed in a centrifuge and centrifuged at a rate of 6000 r / min for 20 min to obtain leaf protein precipitate;

[0044] S6: Freeze drying: The leaf protein precipitate obtained in step S5 is placed in a -60°C freeze dryer and freeze-dried for 12 hours to obtain a finished leaf protein product.

[0045] Example 3

[0046] The present embodiment provides a method for extracting protein from fresh Moringa leaves by adding Lactobacillus plantarum and fermenting, comprising the following steps:

[0047] S1: Pretreatment: Wash the freshly picked Moringa leaves with clean water to remove impurities such as rotten leaves and sand; cut the washed Moringa leaves into small pieces of 1-2 cm for later use;

[0048] S2: Pulping: Put the cut fresh Moringa leaves into a pulper, add water at a solid-liquid ratio of 1:13 and pulp for 3 minutes to obtain freshly squeezed leaf pulp;

[0049] S3: Leaf pulp separation: Use a 300-mesh nylon mesh filter bag to filter the freshly squeezed leaf pulp to separate the leaf residue and green juice; use deionized water to clean the pulper, combine the obtained cleaning liquid with the leaf residue, and filter again with a 300-mesh nylon mesh filter bag to collect all the leaf residue and green juice;

[0050] S4: Fermentation: Add 4×10 Lactobacillus plantarum to the green juice obtained in step S3 6 / mL, stir at room temperature for 3 minutes to mix evenly, and place in a 30℃ constant temperature incubator for anaerobic fermentation to obtain the fermentation liquid;

[0051] S5: centrifugal separation: the fermentation liquid obtained in step S4 is placed in a centrifuge and centrifuged at a rate of 5000 r / min for 15 min to obtain leaf protein precipitate;

[0052] S6: Freeze drying: The leaf protein precipitate obtained in step S5 is placed in a -60°C freeze dryer and freeze-dried for 12 hours to obtain a finished leaf protein product.

[0053] Comparative Example 1

[0054] The difference between this comparative example and Example 3 is that Moringa leaf powder is used instead of Moringa leaf, and other steps remain unchanged.

[0055] Comparative Example 2

[0056] The difference between this comparative example and Example 3 is that in step S2, water is added at a material-liquid ratio of 1:30 for slurrying for 10 minutes, and the other steps remain unchanged.

[0057] Comparative Example 3

[0058] The difference between this comparative example and Example 3 is that in step S4, anaerobic fermentation is carried out in a constant temperature incubator at 30° C. for 10 h.

[0059] Test method:

[0060] p(%)=[(w1×p2) / (w2×p1×p3)]

[0061] Where: p: The percentage of extracted leaf protein in total protein (leaf protein extraction rate)

[0062] w1: dry weight of extracted leaf protein

[0063] w2: The quality of fresh Moringa leaves processed each time

[0064] p1: dry matter content in fresh Moringa samples

[0065] p2: Percentage of crude protein in the dry weight of leaf protein in the extract

[0066] p3: The percentage of crude protein in dry matter in fresh samples

[0067] The collected and extracted Moringa leaf protein concentrate was stored in a -20°C low-temperature refrigerator, and its crude protein content was determined by the Dumas combustion method.

[0068] Leaf protein extraction rate = (grams of protein in leaf protein / grams of protein in fresh sample) × 100%

[0069] Leaf protein yield = (leaf protein dry weight in grams obtained from fresh sample / fresh sample in grams) × 100%

[0070] The test factors and results of each embodiment and comparative example are shown in Table 1.

[0071] Table 1 Performance test results

[0072]

[0073]

[0074] Table 2 Response surface experimental design and results

[0075]

[0076] Table 3 Analysis of variance of regression model

[0077]

[0078]

[0079] Note: * indicates significant difference (P<0.05); ** indicates extremely significant difference (P<0.01).

[0080] Based on the above performance test results and Figure 1 It can be seen that the leaf protein extraction rate and crude protein content of Examples 1-3 are both high; in particular, the comprehensive performance of Example 3 is the most outstanding. However, since Comparative Examples 1-3 do not adopt the necessary technical solutions, their corresponding performance tests are significantly worse than those of the Examples. The above experimental results further prove the importance of the technical solutions defined in the present invention for its technical effects.

[0081] The above is 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 principles 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 fresh Moringa leaf protein by adding Lactobacillus plantarum fermentation, characterized in that: The steps include: S1: Pretreatment: clean fresh Moringa leaves with water, remove impurities, cut into sections for later use; S2: beating: put the fresh Moringa leaves obtained in step S1 into a beating machine, add water and beat to obtain freshly squeezed leaf pulp; S3: Leaf pulp separation: freshly squeezed leaf pulp is filtered to separate leaf residue and green juice; the cleaning liquid from the pulping machine and the leaf residue are mixed and filtered to collect all the leaf residue and green juice; S4: fermentation: adding Lactobacillus plantarum to the green juice obtained in step S3, mixing evenly, placing in a constant temperature incubator for anaerobic fermentation to obtain a fermentation liquid; S5: centrifugal separation: placing the fermentation liquid obtained in step S4 in a centrifuge for centrifugal separation to obtain leaf protein precipitate; S6: Freeze drying: The leaf protein precipitate obtained in step S5 is placed in a freeze dryer for freeze drying to obtain a finished leaf protein product.

2. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, Step S1 is specifically as follows: washing the picked fresh Moringa leaves with clean water to remove impurities such as rotten leaves, mud and sand; cutting the washed fresh Moringa leaves into small segments of 1-2 cm for later use.

3. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, In step S2, the cut fresh Moringa leaves are placed in a pulper, and water is added at a solid-liquid ratio of 1:13 for pulping for 2-3 minutes to obtain freshly squeezed leaf pulp.

4. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, In step S3, a 300-mesh nylon mesh filter bag is used to filter the freshly squeezed leaf pulp to separate leaf residue and green juice; the pulper is cleaned with deionized water, the obtained cleaning liquid is combined with the leaf residue, and filtered again with a 300-mesh nylon mesh filter bag to collect all the leaf residue and green juice.

5. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, In step S4, 4×10 Lactobacillus plantarum was added to the leaf juice. 6 / mL, stir at room temperature for 3min.

6. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, Step S4: Place the mixture in a 30°C constant temperature incubator for anaerobic fermentation for 21 hours.

7. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, In step S5, the fermentation liquid is placed in a centrifuge and centrifuged at a rate of 5000 r / min for 15 minutes to obtain leaf protein precipitation.

8. A method for extracting fresh Moringa leaf protein by adding plant lactobacillus fermentation according to claim 1, characterized in that, In step S5, the leaf protein precipitate is placed in a -60°C freeze dryer for lyophilization for 12 hours to obtain a leaf protein product.

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