Method for preparing ricinus communis meal feed and ricinus communis peptide from ricinus communis meal

Abstract

The invention discloses a method for preparing castor bean meal feed and castor bean peptide from castor bean meal, which comprises the following steps: step 1, taking bacteria for fermentation, and adding water for heat preservation and activation; step 2, adding different enzymes into the activated bacterial liquid according to a certain proportion; step 3, adding the bacterium enzyme compound liquid into the detoxified castor bean meal, and performing heat-preservation fermentation; step 4, drying and crushing the detoxified castor bean meal to obtain fermented detoxified castor bean meal powder; and step five to step ten, extracting castor peptide. By utilizing microbial fermentation and enzymolysis methods, antinutritional factors and harmful substances in the detoxified castor bean meal are reduced, and the nutritional value and safety of the detoxified castor bean meal as feed are improved; when the feed is used for breeding by partially replacing basic ration, the digestibility and growth performance of bred animals can be remarkably improved, meanwhile, the problem of protein resource shortage is relieved, and the feed has important economic and social significance.

Description

Technical Field

[0001] The invention relates to the technical field of enzymatic hydrolysis and fermentation of castor meal, in particular to a method for preparing castor meal feed and ricin peptide by using castor meal. Background Art

[0002] Castor meal contains 37.7% crude protein, 5.40% crude fat, 30.13% crude fiber, 0.51% calcium, 0.54% phosphorus, and is rich in amino acids. However, castor meal obtained after oil extraction also contains a certain amount of toxic substances, such as ricin, ricinine, castor allergen and hemagglutinin. These toxic substances seriously restrict the further development and utilization of the nutrients in castor meal.

[0003] Detoxified castor meal is specially processed by our laboratory to remove toxic and harmful substances such as ricin, but there are still many restrictions on the use of detoxified castor meal in breeding production. As early as 1992, some people reported that when 4% detoxified castor meal was added to the broiler diet, the dry matter metabolism rate, nitrogen retention rate and weekly weight gain in the diet were not significantly different from those in the control group (corn-soybean meal type) (P>0.05). When 13.6% and 23.2% detoxified castor meal were added, the dry matter metabolism rate decreased by 11.65% and 17.08% respectively compared with the control group, and the nitrogen retention rate decreased by 17.76% and 17.55% respectively compared with the control group. The weekly weight gain of the experimental broilers was also lower than that of the control group. In 2014, Ren Chunyan et al. showed that using less than 7.5% detoxified castor meal to replace cake meal as protein feed in the dairy cow diet would basically not affect the rumen fermentation parameters of dairy cows, but would have a certain negative impact on the digestibility of nutrients. Therefore, the current low usage of detoxified castor meal and the anti-nutritional factors that are not conducive to animal growth have greatly restricted the use of detoxified castor meal in animal husbandry and production. Summary of the invention

[0004] The purpose of the present invention is to provide a method for preparing castor meal feed and ricin peptide by using castor meal in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provided is a method for preparing castor meal feed and ricin peptide using castor meal, comprising the following steps:

[0007] Step 1: Take the fermentation bacteria, add water and keep warm to activate them;

[0008] Step 2, adding different enzymes in a certain proportion to the activated bacterial solution in step 1;

[0009] Step 3, adding the bacterial enzyme complex solution obtained in step 2 to the detoxified castor meal, mixing evenly, and performing heat preservation fermentation;

[0010] Step 4, drying the fermented and detoxified castor meal, crushing it to obtain fermented and detoxified castor meal powder, and packaging it to obtain castor meal feed;

[0011] Step 5, adding a certain ratio of water to the fermented detoxified castor meal powder to extract, and letting it stand to collect the supernatant;

[0012] Step 6, passing the supernatant obtained in step 5 through a ceramic membrane and an ultrafiltration membrane;

[0013] Step 7, adding alcohol to the filtrate obtained in step 6, and centrifuging to remove macromolecular proteins;

[0014] Step 8, separating and decolorizing the supernatant obtained in step 7 by passing it through a macroporous resin, and eluting it with distilled water and ethanol in sequence;

[0015] Step nine, separating the eluate containing ricin obtained in step eight through a gel column to obtain a separated solution;

[0016] Step 10, concentrating the separated liquid from step 9 using a rotary evaporator to obtain ricin.

[0017] Furthermore, the fermentation bacteria are Lactobacillus rhamnosus and Candida utilis, and the mass ratio thereof is 1:(1-10).

[0018] Furthermore, the Lactobacillus rhamnosus has an activity of 5×10 11 / g, the activity of the Candida utilis was 5×10 11 / g; the concentration of Lactobacillus rhamnosus in the bacterial solution is 10-12g / L, and the concentration of Candida utilis is 10-12g / L.

[0019] Furthermore, the different types of enzymes include acid protease, cellulase, xylanase and flavor protease, and their mass ratio is 1:(1-5):(1-5):(1-5).

[0020] Furthermore, the acidic protease has an activity of 100,000 U / g, the cellulase has an activity of 100,000 U / g, the xylanase has an activity of 100,000 U / g, and the flavor protease has an activity of 100,000 U / g; the concentrations of the acidic protease, the cellulase, the xylanase, and the flavor protease in the bacterial enzyme complex liquid are 5-9 g / L, 5-9 g / L, 5-9 g / L, and 5-9 g / L.

[0021] Furthermore, in step three, the fermentation temperature is 40-45°C and the fermentation time is 36-48h.

[0022] Furthermore, in the step three, the volume / weight of the bacterial enzyme complex solution and the detoxified castor meal is 1 L / (10-20) kg.

[0023] Furthermore, in step six, after the supernatant passes through a ceramic membrane and an ultrafiltration membrane, components less than 10 kDa are collected.

[0024] Furthermore, in step seven, the alcohol added is 80% ethanol.

[0025] Furthermore, in step nine, the gel column used is a Sephadex G-25 gel column.

[0026] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0027] The present invention utilizes microbial fermentation and enzymatic hydrolysis to reduce anti-nutritional factors and harmful substances in detoxified castor meal, thereby improving the nutritional value and safety of the detoxified castor meal as a feed; using the detoxified castor meal as a partial substitute for basic diet for breeding can significantly improve the digestibility and growth performance of breeding animals, while alleviating the problem of protein resource shortage, and has important economic and social significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The feed-to-egg ratio results of the breeding experiment using different castor meal feed substitutes in Example 3.

[0029] Figure 2 The following are the egg production results of the breeding experiment using different castor meal feed substitutes in Example 3.

[0030] Figure 3 The figures are the mortality rate results of the breeding experiment using different castor meal feeds as substitutes in Example 3.

[0031] Figure 4 The antibacterial effect of ricin peptide in Example 4 is shown. DETAILED DESCRIPTION

[0032] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0033] Example 1

[0034] This embodiment provides a method for preparing castor meal feed and ricin peptide using castor meal, which specifically comprises the following steps:

[0035] Step 1: Take 12g of Lactobacillus rhamnosus and 5×10 11 / g, 12g Candida utilis active 5×10 11 / g was added into 1L sterile water and placed in a 37℃ incubator for activation for 1h;

[0036] Step 2: Take 9 g of acid protease with an activity of 100,000 U / g, cellulase with an activity of 100,000 U / g, xylanase with an activity of 100,000 U / g, and flavor protease with an activity of 100,000 U / g, respectively, and add them to the activated bacterial solution in step 1, and stir to dissolve to prepare a bacterial enzyme complex solution;

[0037] Step 3, adding the bacterial enzyme complex liquid obtained in step 2 to 10 kg of detoxified castor meal, adding water to adjust the moisture content to 40%, stirring and mixing evenly, sealing the bag, and placing it in an incubator at 40° C. for fermentation for 48 hours;

[0038] Step 4, drying the fermented and detoxified castor meal at a temperature of about 90° C. to a moisture content of 6-10%, crushing the fermented and detoxified castor meal powder to obtain the fermented and detoxified castor meal powder, and packaging the fermented and detoxified castor meal powder to obtain castor meal feed; and at the same time, extracting the nutritional components of the fermented and detoxified castor meal powder;

[0039] Step 5: add 6 times volume of water to 1 kg of fermented detoxified castor meal powder, stir with a mixer at 180 r / min for 1 hour, let it stand for 1 hour and take the supernatant;

[0040] Step 6: Pass the supernatant obtained in step 5 through a ceramic membrane and an ultrafiltration membrane to collect components <10 kDa, and place the filtrate in a refrigerator at 4°C for later use;

[0041] Step 7, adding 80% ethanol to the filtrate obtained in step 6 and centrifuging to remove macromolecular proteins, collecting the supernatant by centrifugation, and placing it in a refrigerator at 4°C for later use;

[0042] Step 8, separating and decolorizing the supernatant obtained in step 7 by passing it through a macroporous resin, eluting it with distilled water and 70% ethanol in sequence, and placing the eluate containing ricin in a refrigerator at 4°C for later use;

[0043] Step nine, separating the eluate from step eight through a Sephadex G-25 gel column, using distilled water as the eluent, and collecting different components respectively;

[0044] Step ten, concentrating the eluate from step nine using a rotary evaporator, and further drying to obtain ricin.

[0045] The nutritional components and amino acid composition of castor meal before and after fermentation are shown in Table 1-2:

[0046] Table 1

[0047] project Moisture% Crude protein% Crude fat % Crude fiber % Crude ash % Detoxified castor meal 6.31 54.25 1.47 15.68 9.3 Fermented detoxified castor meal powder 8.02 58.16 1.29 10.27 6.31

[0048] Table 2

[0049]

[0050]

[0051] Example 2

[0052] This embodiment provides a method for preparing castor meal feed using castor meal, which specifically comprises the following steps:

[0053] Step 1: Take 12kg of Lactobacillus rhamnosus and 5×10 11 / g, 12kg Candida utilis active 5×10 11 / g was added into a fermentation tank with 1T sterile water and cultured and activated at 37℃ for 1h;

[0054] Step 2, respectively taking 9 kg of acid protease activity 100,000 U / g, cellulase activity 100,000 U / g, xylanase activity 100,000 U / g, and flavor protease activity 100,000 U / g and adding them into the fermentation tank of step 1, stirring and dissolving to prepare a bacterial enzyme complex liquid;

[0055] Step 3, adding the bacterial enzyme complex liquid obtained in step 2 to 10T of detoxified castor meal, adding water to adjust the moisture content to 40%, stirring and mixing evenly, putting into a fermentation tank, and fermenting at 40°C for 48h;

[0056] Step 4: drying the fermented and detoxified castor meal at a temperature of about 90° C. to a moisture content of 6-10%, crushing the fermented and detoxified castor meal powder to obtain the castor meal feed.

[0057] Example 3: Farming test

[0058] The castor meal feed prepared in Example 2 was applied to a large-scale breeding farm in Dongtai City, Jiangsu Province for breeding experiments, and the specific implementation method is as follows:

[0059] 1500 Roman brown laying hens with an age of 300 days in the laying period were selected, and the test period was 30 days. The 1500 laying hens were divided into 5 groups, each group had 3 replicates, and each replicate had 100 laying hens. The control group 1 used a basic diet formula of 66.5% corn, 25% soybean meal, 7.5% lime powder, 0.7% soybean oil, and 0.3% salt. The control group 2 used the castor meal feed in Example 2 to completely replace all the soybean meals in the basic diet formula. The test group 1 used the castor meal feed to replace 20% of all the soybean meals in the basic diet. The test group 2 used the castor meal feed to replace 40% of all the soybean meals in the basic diet. The test group 3 used the castor meal feed to replace 60% of all the soybean meals in the basic diet.

[0060] Feed according to the average feed intake of the farm, 95g per chicken per day on average, follow the feeding, drinking water and egg picking methods of the farm, calculate the number of eggs and total egg weight of each group per day, analyze the feed-egg ratio of different groups, count the number of deaths and calculate the death rate. The test results are as follows:

[0061] Depend on Figure 1 It can be seen that the feed-to-egg ratio of control group 2 is low, showing a fluctuating trend over time, and the lowest feed-to-egg ratio is about 1.7. The feed-to-egg ratio of experimental group 3 is also low, first showing a downward trend and finally rising in the fourth week. There is little difference in the feed-to-egg ratio between control group 1 and experimental groups 1 and 2 in the first two weeks. The feed-to-egg ratio of experimental group 2 gradually decreases in the third week, and the feed-to-egg ratio of control group 1 begins to increase in the third week and finally reaches about 2.01. The results of this experiment show that castor meal replacing soybean meal is beneficial to reducing the feed-to-egg ratio, and it is also beneficial to improving the feed utilization efficiency of laying hens.

[0062] Depend on Figure 2 It can be seen that the egg production rate of each group showed an upward trend. The control group 2 showed better egg production performance, which showed a gradually increasing trend over time, and the final egg production rate was 88.7%. The egg production rates of the control group 1 and the experimental group 1 were not much different in the first to third weeks. The egg production rate of the control group 1 was higher than that of the experimental group 1. From the third to fourth week, the egg production rate of the experimental group 1 was higher than that of the control group 1. This experimental result shows that castor meal can replace soybean meal to improve the egg production performance of laying hens.

[0063] Depend on Figure 3 It can be seen that the mortality rate of control group 1 was the highest, the mortality rate of control group 2 was the lowest, and the mortality rate of experimental groups 1-3 gradually decreased, indicating that castor meal feed can reduce the mortality rate of laying hens, and replacing soybean meal will not affect the health of laying hens.

[0064] Example 4

[0065] The ricin peptide prepared in Example 1 was subjected to a punching method antibacterial test, and the specific implementation method is as follows:

[0066] 1. Bacterial source and culture medium

[0067] Indicator bacteria: Escherichia coli K99, which was donated by Zhejiang University. Escherichia coli is the most common pathogenic bacteria, so Escherichia coli was selected as the indicator.

[0068] LB medium: peptone 10 g / L, yeast extract 5 g / L, sodium chloride 10 g / L, pH 7.3; 1.5% agar was added to the solid medium.

[0069] 2. Sample preparation

[0070] Preparation of test samples: 1g of ricin peptide was placed in a 50ml centrifuge tube, 20ml of sterile water was added, and the solution was shaken on a vortex mixer for 3-5min. The solution was filtered through a 0.22μm filter membrane, and the filtrate was diluted 2-fold with sterile water to obtain 60000μg / mL. The solution was further diluted 2-fold with the extract to prepare solutions with concentrations of 50000, 25000, 12500, 6250, 3125, 1562.5, 781.25, 390.63, 195.31, 97.66, 48.83, 24.41, and 12.21μg / mL, and stored in a 4℃ refrigerator for later use.

[0071] 3. Preparation of indicator bacteria solution

[0072] Preparation of bacterial solution: Pick a single colony of Escherichia coli and inoculate it into 9mL LB liquid culture medium, and culture it at 37℃ with constant temperature shaking for 8h; take 1mL of bacterial solution into 9mL LB liquid culture medium, shake it thoroughly, then take 1ml of dilution solution and add it to 9mL LB liquid culture medium, shake it thoroughly, and place it in a 4℃ refrigerator for later use.

[0073] 4. Experimental steps

[0074] Heat and melt the LB solid medium, let it cool to about 60°C, add 15 mL of the medium to each plate, and let it cool and solidify;

[0075] Take 100ul of indicator bacteria from each plate and spread it evenly;

[0076] Use a sterilized 9mm diameter puncher to punch holes in the agar and add the sample to be tested; each sample is repeated three times, and sterile water is used as a control;

[0077] Place the plate in a 4°C refrigerator for more than 2 h, and then culture it in a 37°C biochemical incubator;

[0078] Observe and record the diameter of the inhibition zone.

[0079] 5. Test results

[0080] like Figure 4 , Serial numbers 1, 2, 3, 4, 5, 6, and 7 represent 390.63, 195.31, 97.66, 48.83, 24.41, 12.21 μg / ml and control, respectively. The minimum inhibitory concentration of ricinol after 16 h of culture was 97.66 μg / ml, and the diameter of the inhibition zone was 14 mm.

[0081] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing castor meal feed and ricin using castor meal, characterized in that: The steps include: Step 1: Take the fermentation bacteria, add water and keep warm to activate them; Step 2, adding different enzymes in a certain proportion to the activated bacterial solution in step 1; Step 3, adding the bacterial enzyme complex solution obtained in step 2 to the detoxified castor meal, mixing evenly, and performing heat preservation fermentation; Step 4, drying the fermented and detoxified castor meal, crushing it to obtain fermented and detoxified castor meal powder, and packaging it to obtain castor meal feed; Step 5, adding a certain ratio of water to the fermented detoxified castor meal powder to extract, and letting it stand to collect the supernatant; Step 6, passing the supernatant obtained in step 5 through a ceramic membrane and an ultrafiltration membrane; Step 7, adding alcohol to the filtrate obtained in step 6, and centrifuging to remove macromolecular proteins; Step 8, separating and decolorizing the supernatant obtained in step 7 by passing it through a macroporous resin, and eluting it with distilled water and ethanol in sequence; Step nine, separating the eluate containing ricin obtained in step eight through a gel column to obtain a separated solution; Step 10, concentrating the separated liquid from step 9 using a rotary evaporator to obtain ricin.

2. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: The fermentation bacteria are Lactobacillus rhamnosus and Candida utilis, and the mass ratio thereof is 1:(1-10).

3. The method for preparing castor meal feed and ricin using castor meal according to claim 2, characterized in that: The activity of Lactobacillus rhamnosus was 5×10 11 / g, the activity of the Candida utilis was 5×10 11 / g; the concentration of Lactobacillus rhamnosus in the bacterial solution is 10-12g / L, and the concentration of Candida utilis is 10-12g / L.

4. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: The different types of enzymes include acid protease, cellulase, xylanase and flavor protease, and the mass ratio thereof is 1:(1-5):(1-5):(1-5).

5. The method for preparing castor meal feed and ricin using castor meal according to claim 4, characterized in that: The acidic protease has an activity of 100,000 U / g, the cellulase has an activity of 100,000 U / g, the xylanase has an activity of 100,000 U / g, and the flavor protease has an activity of 100,000 U / g; the concentrations of the acidic protease, the cellulase, the xylanase, and the flavor protease in the bacterial enzyme complex liquid are 5-9 g / L, 5-9 g / L, 5-9 g / L, and 5-9 g / L.

6. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: In the step 3, the fermentation temperature is 40-45°C and the fermentation time is 36-48h.

7. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: In the step 3, the volume / weight of the bacterial enzyme complex solution and the detoxified castor meal is 1 L / (10-20) kg.

8. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: In step six, after the supernatant passes through a ceramic membrane and an ultrafiltration membrane, components <10 kDa are collected.

9. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: In the step seven, the alcohol added is 80% ethanol.

10. The method for preparing castor meal feed and ricin using castor meal according to claim 1, characterized in that: In the step nine, the gel column used is a Sephadex G-25 gel column.

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