A mulberry leaf fermentation method based on electrically stimulating bacteria, and products and applications thereof

Abstract

The application discloses a mulberry leaf fermentation method based on electric stimulation of bacteria and products and application thereof, and the method comprises the following steps: dissolving dry mulberry leaf powder with water, adding a mixed bacterial population of electrically stimulated Bacillus cereus and Lactobacillus casei plantarum, and performing fermentation culture under the condition of electric stimulation. The fermentation mulberry leaf powder obtained by optimizing different probiotic bacterial species combinations and applying electric stimulation to the fermentation process has not only improved protein content but also greatly reduced anti-nutritional factors phytic acid and tannin, and has obviously improved nutritional value. Compared with other non-fermented mulberry leaves and other bacterial agent fermented mulberry leaves, the fermentation method has obvious advantages, and provides a new method for effective utilization of Chinese silkworm and mulberry resources in China.

Description

TECHNICAL FIELD

[0001] The present application relates to a mulberry leaf fermentation method based on electrically stimulating bacteria, and products and applications thereof, and belongs to the field of agricultural technology. BACKGROUND

[0002] China is short of feed resources, and the gap between supply and demand is large. Through microbial fermentation and other technologies, the quality of traditional feed grains can be improved, and non-traditional feed resources can be developed. China is a large country of traditional mulberry cultivation and silkworm breeding. Mulberry leaves have high nutritional value and are a new type of feed resource with good development potential. Mulberry leaves, which have high nutritional value, are a high-quality and complete feed. Compared with alfalfa, which is known as the "king of feed", mulberry leaves have higher nutritional value and stronger health care effect. When applied in animal breeding, mulberry leaves can improve production performance, increase immunity, and improve the quality of livestock products, and reduce feeding costs. Mulberry leaves contain bioactive substances such as flavonoids, polyphenols, polysaccharides, and alkaloids, which have antioxidant, antibacterial, and immune-enhancing effects, and have health care functions for livestock and poultry. However, the presence of anti-nutritional factors such as phytic acid and tannin in mulberry leaves limits the amount of mulberry leaves used in feed. Microbial fermentation can effectively reduce the content of anti-nutritional factors and improve the utilization rate of mulberry leaves. Therefore, the application of fermented mulberry leaves in animal feed can not only alleviate the problem of feed shortage, but also improve the meat quality and immunity of animals, which is conducive to the sustainable development of animal husbandry.

[0003] Currently, there are many studies on mulberry leaf fermentation methods. However, there are still few fermentation preparation methods for reducing the anti-nutritional factors of mulberry leaves. Electric stimulation is a method of promoting cell proliferation and accelerating microbial metabolism by applying a small voltage or current. Compared with general electrochemical methods, it has the advantages of low power consumption and no production of toxic and harmful substances. Moreover, electric stimulation technology does not require the addition of ion exchange membranes and has low requirements for electrode materials, so it has the advantages of simple reactor and easy operation. However, there are few reports on the use of electric stimulation technology to improve the anti-nutritional factors of fermented mulberry leaves. SUMMARY

[0004] The technical problem to be solved by the present application is to provide a method for increasing the crude protein content of mulberry leaves while reducing the content of anti-nutritional factors phytic acid and tannin, which is low in price and simple in process.

[0005] The technical problem to be solved by the present application is to provide a method for increasing the crude protein content of mulberry leaves while reducing the content of anti-nutritional factors phytic acid and tannin, which is low in price and simple in process.

[0006] Technical solution: To solve the above technical problems, the present application provides a mulberry leaf fermentation method based on electric stimulation of bacteria, comprising the following steps: dissolving dry mulberry leaf powder with water, adding mixed bacterial flora of electrically stimulated Bacillus cereus and Lactobacillus casei plantarum, and carrying out fermentation culture under the condition of electric stimulation to obtain the low anti-nutritional factor mulberry leaf product.

[0007] Among them, the mass ratio of Bacillus cereus and Lactobacillus casei plantarum is 1:1-2.

[0008] Among them, the current of the electric stimulation in the electrically stimulated Bacillus cereus is 5-25mA.

[0009] Among them, the current of the electric stimulation is 10-25mA.

[0010] Among them, the inoculation amount of the mixed bacteria of Bacillus cereus and Lactobacillus casei plantarum is 5-10%.

[0011] Among them, the mass ratio of the mulberry leaf powder and water is 5-10:100.

[0012] Among them, the fermentation culture time is 48-72h, and the temperature is 25-37℃.

[0013] The present application also provides a low anti-nutritional factor mulberry leaf product prepared by the method.

[0014] The present application also provides a feed comprising the low anti-nutritional factor mulberry leaf product.

[0015] The present application also provides the application of the low anti-nutritional factor mulberry leaf product in the preparation of poultry, aquatic feed.

[0016] The present application aims to improve the microbial fermentation method, and decompose the protein and other difficult-to-digest macromolecules in mulberry leaves into small-molecule nutrients that can be easily absorbed by the body with the help of microorganisms. In particular, the fermentation of tannin, phytic acid and other anti-nutritional factors in mulberry leaves is optimized for digestion, thereby improving the palatability and nutritional value of fermented mulberry leaves, and better applying mulberry leaf powder in animal breeding.

[0017] Beneficial effects: Compared with the prior art, the present application has the following remarkable advantages: the present application applies electric stimulation to the liquid fermentation of feed mulberry leaf powder, the obtained fermented mulberry leaf powder not only has increased protein content, but also has 45%-60% degradation of anti-nutritional factor phytic acid and 40%-70% degradation of tannin in the fermented mulberry leaf powder, and the nutritional value is significantly higher than that of unfermented mulberry leaf powder; and through freeze-drying, a large amount of active probiotics are retained, so that the absorption of nutrients by animals can be improved, the growth performance and disease resistance of animals can be improved, the feed input cost can be reduced, and the economic benefit of breeding can be improved. Mulberry leaves and other leaf raw materials have wide sources and low prices, the preparation process of fermented mulberry leaves is simple, and the application of fermented mulberry leaves as an effective plant protein in feed provides a new direction for the effective utilization of China's mulberry resources. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 Changes of tannase activity of Bacillus cereus after electric stimulation. DETAILED DESCRIPTION

[0019] The technical solutions of the present application will be further described below in combination with the drawings.

[0020] The bacterial culture medium involved in the present application is beef extract protein peptone medium, and the formula of the beef extract protein peptone liquid medium is (g / L): beef extract 3; NaCl 5; protein peptone 10; pH 7.0-7.2. Sterilization is performed at 121℃ for 20 min.

[0021] The Bacillus cereus used for mulberry leaf fermentation is from China Microbial Strain Query Network, and the item number is Bio-67713; the Lactobacillus casei plantarum is from Shandong Zhongke Jiayi Biological Engineering Co., Ltd., and the item number is JYLP-326.

[0022] Example 1: Experiment of electric stimulation enhancing tannase activity of Bacillus cereus

[0023] Beef extract protein peptone liquid medium is prepared, and the electrically stimulated culture bottle of Bacillus cereus, electrode material and culture medium are sterilized by high-pressure steam (sterilization is performed at 0.11Mpa, 121℃ for 20 min) before the experiment. Bacillus cereus is inoculated at an inoculation amount of 1%: 3mL of uniformly oscillated bacterial suspension is taken and inoculated into two 500mL culture bottles containing 300mL of liquid medium, so that the concentration of the culture solution is 1×10 6 cfu / mL, wherein one culture bottle is not electrified (L), and the other culture bottle is subjected to 5mA direct current stimulation (EL) by using a platinum electrode (electrode spacing 2cm). The culture bottles are placed in a 37℃ constant temperature shaker (150r / min) for 48h. OD 600The data was recorded. The tannase activity of Bacillus cereus was analyzed according to the determination method of tannase activity, and the results showed that the 5mA electric stimulation improved the tannase production characteristics of Bacillus cereus, and the enzyme activity increased from 0.202U to 0.309U Figure 1 ).

[0024] Example 2 Fermentation of mulberry leaf powder

[0025] A liquid fermentation method of mulberry leaf powder comprises the following steps:

[0026] (1) The freshly picked mulberry leaves are wilted and dried, then put into a pulverizer for 2 minutes to obtain mulberry leaf powder;

[0027] (2) 90 mL of sterile water is added to 10 g of the mulberry leaf powder, and mixed uniformly to obtain fermentation base raw material;

[0028] (3) The mixed bacteria of 5mA electric stimulation Bacillus cereus and Lactobacillus plantarum are added to the fermentation base raw material, and mixed uniformly, then fermented at 25mA electric stimulation and 35°C for 48h; wherein the weight ratio of Bacillus cereus and Lactobacillus plantarum is 1:1; the inoculation amount of the mixed bacteria is 5%. The control is unfermented mulberry leaf powder. The results in Table 1 show that the nutritional structure of the fermented mulberry leaf powder is significantly improved, the content of crude protein is increased, the pH of the mulberry leaf powder is reduced, and the degradation rates of the anti-nutritional factors tannin and phytic acid are 66.9% and 56.6% respectively.

[0029] Table 1 Change of nutritional components of mulberry leaf before and after fermentation

[0030]

[0031] Example 3

[0032] A liquid fermentation method of mulberry leaf powder comprises the following steps:

[0033] (1) The freshly picked mulberry leaves are wilted and dried, then put into a pulverizer for 3 minutes to obtain mulberry leaf powder;

[0034] (2) 100 mL of sterile water is added to 5 g of the mulberry leaf powder, and mixed uniformly to obtain fermentation base raw material;

[0035] (3) The mixed bacteria of 15mA electric stimulation Bacillus cereus and Lactobacillus plantarum are added to the fermentation base raw material, and mixed uniformly, then fermented at 20mA electric stimulation and 25°C for 72h; wherein the weight ratio of Bacillus cereus and Lactobacillus plantarum is 1:2; the inoculation amount of the mixed bacteria is 10%.

[0036] Example 4

[0037] A liquid fermentation method of mulberry leaf powder steps:

[0038] (1) The freshly picked mulberry leaves are wilted and dried, then put into a pulverizer for 5 minutes to obtain mulberry leaf powder;

[0039] (2) 100 mL of sterile water is added to 7 g of the mulberry leaf powder, and mixed evenly to obtain fermentation base raw materials;

[0040] (3) The mixed bacteria of Bacillus cereus and Lactobacillus plantarum after 25 mA electric stimulation are added to the fermentation base raw materials, and mixed evenly. After 60 h of fermentation at 10 mA electric stimulation and 37°C, the weight ratio of Bacillus cereus and Lactobacillus plantarum is 1:2; the inoculation amount of the mixed bacteria is 7%. The results in Table 2 show that the nutritional structure of the mulberry leaf powder after liquid fermentation is significantly improved, the content of crude protein is increased, and the pH of the mulberry leaf powder is reduced. The degradation rates of the anti-nutritional factor tannin are 69.7% and 68.8%, respectively; the degradation rates of phytic acid are 60.4% and 59.2%, respectively.

[0041] Table 2 Nutritional component changes of mulberry leaf powder prepared in Examples 3 and 4

[0042]

[0043] Comparative Example 1

[0044] A liquid fermentation method of mulberry leaf powder steps:

[0045] (1) The freshly picked mulberry leaves are wilted and dried, then put into a pulverizer for 2 minutes to obtain mulberry leaf powder;

[0046] (2) 90 mL of sterile water is added to 10 g of the mulberry leaf powder, and mixed evenly to obtain fermentation base raw materials;

[0047] (3) The Bacillus cereus after electric stimulation is added to the fermentation base raw materials, and mixed evenly. After 48 h of fermentation at 25 mA electric stimulation and 35°C, the inoculation amount of the bacteria solution is 5%.

[0048] Comparative Example 2

[0049] A liquid fermentation method of mulberry leaf powder steps:

[0050] (1) The freshly picked mulberry leaves are wilted and dried, then put into a pulverizer for 2 minutes to obtain mulberry leaf powder;

[0051] (2) 90 mL of sterile water is added to 10 g of the mulberry leaf powder, and mixed evenly to obtain fermentation base raw materials;

[0052] (3) adding Bacillus cereus and Lactobacillus plantarum to the fermentation base raw material, mixing uniformly, and then fermenting at 25 mA electric stimulation and 35 °C for 48 h; the inoculation amount of the mixed bacteria is 5%.

[0053] Comparative Example 3

[0054] A liquid fermentation method step of mulberry leaf powder:

[0055] (1) drying the newly picked mulberry leaves after wilting treatment, crushing them in a crusher for 2 min to obtain mulberry leaf powder;

[0056] (2) adding 90 mL of sterile water to 10 g of the mulberry leaf powder, mixing uniformly to obtain a fermentation base raw material;

[0057] (3) adding Bacillus cereus and Lactobacillus plantarum mixed bacteria after electric stimulation to the fermentation base raw material, mixing uniformly, and then fermenting at 35 °C for 48 h; the weight ratio of Bacillus cereus and Lactobacillus plantarum is 1:1; the inoculation amount of the mixed bacteria is 5%.

[0058] Comparative Example 4

[0059] A liquid fermentation method step of mulberry leaf powder:

[0060] (1) drying the newly picked mulberry leaves after wilting treatment, crushing them in a crusher for 2 min to obtain mulberry leaf powder;

[0061] (2) adding 90 mL of sterile water to 10 g of the mulberry leaf powder, mixing uniformly to obtain a fermentation base raw material;

[0062] (3) adding Bacillus cereus and Lactobacillus plantarum mixed bacteria after electric stimulation to the fermentation base raw material, mixing uniformly, and then fermenting at 25 mA electric stimulation and 35 °C for 48 h; the weight ratio of Bacillus cereus and Lactobacillus plantarum is 2:1; the inoculation amount of the mixed bacteria is 5%.

[0063] Comparative Example 5

[0064] A liquid fermentation method step of mulberry leaf powder:

[0065] (1) drying the newly picked mulberry leaves after wilting treatment, crushing them in a crusher for 2 min to obtain mulberry leaf powder;

[0066] (2) adding 90 mL of sterile water to 10 g of the mulberry leaf powder, mixing uniformly to obtain a fermentation base raw material;

[0067] (3) adding the mixed bacteria of Bacillus cereus and Lactobacillus paracasei subjected to electric stimulation into the fermentation base material, mixing uniformly, and then fermenting for 48h under the condition of 30mA electric stimulation and 35℃ temperature; wherein the weight ratio of Bacillus cereus and Lactobacillus paracasei is 1:1; and the inoculation amount of the mixed bacteria is 5%.

[0068] Comparative Example 6

[0069] A liquid fermentation method of mulberry leaf powder comprises the following steps:

[0070] (1) drying the newly picked mulberry leaves after wilting treatment, and crushing them in a pulverizer for 2min to obtain mulberry leaf powder;

[0071] (2) adding 90mL sterile water into 10g of the mulberry leaf powder to obtain fermentation base material;

[0072] (3) adding the mixed bacteria of Bacillus cereus and Lactobacillus paracasei subjected to electric stimulation into the fermentation base material, mixing uniformly, and then fermenting for 48h under the condition of 25mA electric stimulation and 25℃ temperature; wherein the weight ratio of Bacillus cereus and Lactobacillus paracasei is 1:1; and the inoculation amount of the mixed bacteria is 2%.

[0073] Comparative Example 7

[0074] A liquid fermentation method of mulberry leaf powder comprises the following steps:

[0075] (1) drying the newly picked mulberry leaves after wilting treatment, and crushing them in a pulverizer for 2min to obtain mulberry leaf powder;

[0076] (2) adding 90mL sterile water into 10g of the mulberry leaf powder to obtain fermentation base material;

[0077] (3) adding the mixed bacteria of Bacillus cereus and Lactobacillus paracasei subjected to electric stimulation into the fermentation base material, mixing uniformly, and then fermenting for 48h under the condition of 25mA electric stimulation and 25℃ temperature; wherein the weight ratio of Bacillus cereus and Lactobacillus paracasei is 1:1; and the inoculation amount of the mixed bacteria is 2%.

[0078] The experimental results of the comparative examples are shown in Table 3:

[0079] Table 3 Change of nutritional components of mulberry leaves before and after fermentation

[0080]

[0081] As can be seen from Comparative Examples 2-4 and Comparative Examples 1-7, the fermentation method of the present application can greatly increase the protein content of mulberry leaves and reduce the content of anti-nutritional factors tannin and phytic acid affecting the absorption of nutritional substances of mulberry leaves, thereby providing technical support for efficient utilization of mulberry leaf resources.

Claims

1. A mulberry leaf fermentation method based on electrically stimulating bacteria, characterized by, comprising the following steps: Dissolve dry mulberry leaf powder with water, add mixed bacteria of Bacillus cereus ( Bacillus cereus ) and Lactobacillus paracasei ( Lactobacillus casei plantarum ) after electric stimulation, and ferment under the condition of electric stimulation to obtain low anti-nutritional factor mulberry leaf product; the mass ratio of Bacillus cereus ( Bacillus cereus ) and Lactobacillus paracasei ( Lactobacillus casei plantarum ) is 1:1-2; the inoculation amount of mixed bacteria of Bacillus cereus ( Bacillus cereus ) and Lactobacillus paracasei ( Lactobacillus casei plantarum ) is 5-10%; the current of electric stimulation is 5-25 mA; and the mass ratio of mulberry leaf powder and water is 5-10:

100.

2. The method of claim 1, wherein, The current of the electric stimulation is 10-25 mA.

3. The method of claim 1, wherein, The time of the fermentation culture is 48-72 h, and the temperature is 25-37℃.

4. A low-anti-nutritional factor mulberry leaf product prepared by the method of any one of claims 1-3.

5. A feed, characterized in that, The low-anti-nutritional factor mulberry leaf product of claim 4.

6. Use of the low-anti-nutritional factor mulberry leaf product of claim 4 in the preparation of poultry, livestock, and aquatic feed.

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