Method for preparing biological feed by fermenting desiccated coconut with multiple strains
阅读说明：本技术 一种多菌种发酵椰蓉制备生物饲料的方法 (Method for preparing biological feed by fermenting desiccated coconut with multiple strains ) 是由 王学梅 张晓梦 王闫宇 吴军龙 白国松 刘晓烜 孙楠 陈应强 杨雨辉 张昌和 于 2020-06-11 设计创作，主要内容包括：本发明涉及生物饲料技术领域,特别涉及一种多菌种发酵椰蓉制备生物饲料的方法。该方法包括：将椰蓉和豆粕的混合物作为发酵底物,采用酿酒酵母菌、枯草芽孢杆菌组成混合菌种进行有氧发酵2～5天,再添加植物乳杆菌对发酵底物进行厌氧发酵5～15天,通过两步固态发酵从而得到发酵椰蓉生物饲料。三种菌种的活菌数比例为(27～54):(1.9～5.7):36。本发明发酵饲料具有发酵香味,因此能提高畜禽的采食量。通过微生物发酵技术开发海南当地特色的地源性饲料,另外海南属于热带地区,微生物种类繁多,气候适合微生物生长,代谢能力强,能在短时间内能把大量的地源性农业废弃物转化为有用的发酵饲料,从而减少生产开发成本。(The invention relates to the technical field of biological feed, in particular to a method for preparing biological feed by fermenting desiccated coconut with multiple strains. The method comprises the following steps: taking a mixture of the desiccated coconut and the bean pulp as a fermentation substrate, adopting saccharomyces cerevisiae and bacillus subtilis to form a mixed strain for aerobic fermentation for 2-5 days, adding lactobacillus plantarum to carry out anaerobic fermentation on the fermentation substrate for 5-15 days, and carrying out two-step solid state fermentation to obtain the fermented desiccated coconut biological feed. The ratio of viable count of the three strains is (27-54): 1.9-5.7): 36. The fermented feed has the fermented fragrance, so that the feed intake of livestock and poultry can be improved. The method is characterized in that the local characteristic ground source feed of Hainan is developed through a microbial fermentation technology, and Hainan belongs to tropical regions, so that the variety of microorganisms is various, the climate is suitable for the growth of the microorganisms, the metabolic capability is strong, a large amount of ground source agricultural wastes can be converted into useful fermented feed in a short time, and the production and development cost is reduced.)
1. The preparation method of the desiccated coconut feed is characterized by comprising the following steps of: taking the desiccated coconut and the bean pulp as fermentation substrates, firstly adopting a mixed strain consisting of saccharomyces cerevisiae and bacillus subtilis to perform aerobic fermentation on the fermentation substrates for 2-5 days, then adding lactobacillus plantarum to perform anaerobic fermentation for 5-15 days, and obtaining the fermented desiccated coconut biological feed through two-step solid state fermentation;
the volume ratio of the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum is (27-54): 1.9-5.7): 36.
2. The method according to claim 1, wherein the aerobic fermentation is performed for 3 days, and the anaerobic fermentation is performed for 10 days.
3. The method according to claim 1, wherein the ratio of saccharomyces cerevisiae, bacillus subtilis and lactobacillus plantarum is 54:3.8:36 by volume.
4. The method according to claim 1, wherein the concentration of the Saccharomyces cerevisiae is 2.7 × 108CFU/mL, the bacterial liquid concentration of the bacillus subtilis is 1.9 × 107CFU/mL, the concentration of the lactobacillus plantarum bacterial liquid is 3.6 × 108CFU/mL。
5. The preparation method according to claim 4, wherein the addition amount of Saccharomyces cerevisiae, Bacillus subtilis and Lactobacillus plantarum in the fermentation substrate is 6-10% by mass percentage.
6. The method according to claim 5, wherein the fermentation substrate contains Saccharomyces cerevisiae, Bacillus subtilis, and Lactobacillus plantarum in an amount of 10% by mass.
7. The method according to claim 1, wherein the water content of the aerobic fermentation is 36 to 40% by mass.
8. The preparation method according to claim 1, wherein the mass ratio of the desiccated coconut to the soybean meal in the fermentation substrate is (1-2): (1-2).
9. The method for preparing saccharomyces cerevisiae according to any one of claims 1 to 8, wherein the saccharomyces cerevisiae is activated saccharomyces cerevisiae, and the method for activating saccharomyces cerevisiae is as follows: inoculating saccharomyces cerevisiae into a YPD liquid culture medium, culturing for 8-12 hours at 30 ℃, then inoculating into the YPD liquid culture medium again, and culturing for 12-16 hours at 30 ℃ and 150 r/min;
the bacillus subtilis is activated bacillus subtilis, and the activation method of the bacillus subtilis comprises the following steps: inoculating the bacillus subtilis into an LB liquid culture medium, culturing for 8-12 hours at 37 ℃, then inoculating into the LB liquid culture medium again, and culturing for 12-16 hours at 37 ℃ and 150 r/min;
the lactobacillus plantarum is activated lactobacillus plantarum, and the activating method of the lactobacillus plantarum comprises the following steps: and (3) putting lactobacillus plantarum in MRS broth, performing anaerobic culture at 37 ℃ for 8-12 hours, inoculating the lactobacillus plantarum in the MRS broth again, and performing anaerobic overnight culture at 37 ℃ for 12-16 hours.
10. Desiccated coconut feed produced by the production method according to any one of claims 1 to 9.
Technical Field
The invention relates to the technical field of biological feed, in particular to a method for preparing biological feed by fermenting desiccated coconut with multiple strains.
Background
In recent years, the concentrated feed resource is lack and expensive, the abundant coarse feed resource is abandoned or burnt because the concentrated feed resource is not reasonably applied to the feed, and the feed resource is deficient, thereby influencing the development of animal husbandry. The development of the microbial fermented feed industry is one of the important approaches for solving the feed shortage, and simultaneously, the development of ground source resources becomes a research hotspot. The ground source fermented feed has unique nutritive value and good feed quality, can reduce cost through reasonable fermentation, improve the economic benefit of local farms and reduce environmental pollution, and the development of the ground source fermented feed is beneficial to realizing ecological cycle and healthy culture.
The Hainan coconut has rich resources, and the desiccated coconut is used in most foods as a ground source resource with characteristics of Hainan, and is not deeply researched in feed. The desiccated coconut contains a lot of active substances, especially has high fatty acid content, and has great feed development and application potential and high economic value, so that the production of livestock and poultry feed by microbial fermentation of desiccated coconut is beneficial to the development and utilization of Hainan feed resources.
At present, no technology for preparing biological feed by using desiccated coconut exists.
Disclosure of Invention
In view of the above, the invention provides a method for preparing biological feed by fermenting desiccated coconut with multiple strains. The method utilizes the compound microorganisms to prepare the novel high-quality biological feed with rich nutrition, good palatability and high viable bacteria content by two-step solid fermentation of the desiccated coconut, thereby reducing the use of feed antibiotic additives and environmental pollution, ensuring the safety of animal food, and providing certain theoretical basis and practical basis for the development and utilization of desiccated coconut in Hainan province.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a preparation method of desiccated coconut feed, which comprises the following steps: taking a mixture of the desiccated coconut and the bean pulp as a fermentation substrate, firstly adopting a mixed strain consisting of saccharomyces cerevisiae and bacillus subtilis to perform aerobic fermentation on the fermentation substrate for 2-5 days, then adding lactobacillus plantarum to perform anaerobic fermentation for 5-15 days, and obtaining the fermented desiccated coconut biological feed through two-step solid state fermentation;
the volume ratio of the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum is (27-54): 1.9-5.7): 36.
Preferably, the fermentation is carried out aerobically for 3 days and the anaerobic fermentation is carried out for 10 days.
Preferably, the volume ratio of the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum is 54:3.8: 36.
Preferably, the concentration of the saccharomyces cerevisiae liquid is 2.7 × 108CFU/mL, the concentration of the bacillus subtilis liquid is 1.9 × 107CFU/mL, the concentration of the lactobacillus plantarum bacterial liquid is 3.6 × 108CFU/mL。
Preferably, the addition amount of the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum in the fermentation substrate is 6-10% by mass percentage.
Preferably, the addition amount of the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum in the fermentation substrate is 10% by mass percentage.
Preferably, the water content of the aerobic fermentation is 36 to 40% by mass.
Preferably, the water content of the aerobic fermentation is 36% by mass percentage.
Preferably, the mass ratio of the desiccated coconut (fresh sample) to the soybean meal in the fermentation substrate is (1-2): (1-2).
Preferably, the mass ratio of the desiccated coconut (fresh sample) to the soybean meal in the fermentation substrate is 1: 1.
preferably, the saccharomyces cerevisiae is activated saccharomyces cerevisiae, and the activation method of the saccharomyces cerevisiae comprises the following steps: inoculating saccharomyces cerevisiae into a YPD liquid culture medium, culturing for 8-12 hours at 30 ℃, then inoculating into the YPD liquid culture medium again, and culturing for 12-16 hours at 30 ℃ and 150 r/min.
Preferably, the bacillus subtilis is activated bacillus subtilis, and the activating method of the bacillus subtilis comprises the following steps: inoculating the bacillus subtilis into an LB liquid culture medium, culturing for 8-12 hours at 37 ℃, then inoculating into the LB liquid culture medium again, and culturing for 12-16 hours at 37 ℃ and 150 r/min.
Preferably, the lactobacillus plantarum is activated lactobacillus plantarum, and the lactobacillus plantarum activation method comprises the following steps: and (3) putting lactobacillus plantarum in MRS broth, performing anaerobic culture at 37 ℃ for 8-12 hours, inoculating the lactobacillus plantarum in the MRS broth again, and performing anaerobic overnight culture at 37 ℃ for 12-16 hours.
The invention also provides the desiccated coconut feed prepared by the method.
The invention provides a method for preparing biological feed by fermenting desiccated coconut with multiple strains and desiccated coconut feed. The method comprises the following steps: taking a mixture of the desiccated coconut and the soybean meal as a fermentation substrate, carrying out aerobic fermentation for 2-5 days by adopting saccharomyces cerevisiae and bacillus subtilis composite strains, and then adding lactobacillus plantarum to carry out anaerobic fermentation for 5-15 days on the fermentation substrate, thereby obtaining the desiccated coconut fermented feed. The ratio of the viable count of the three bacteria is (27-54): 1.9-5.7): 36. The development of the fermented feed of the local resources in Hainan has the following advantages:
the fermented feed has good fermentation fragrance, so that the feed intake of livestock and poultry can be improved.
Secondly, the local characteristic ground source feed of Hainan is developed and utilized through a fermentation technology, Hainan belongs to tropical regions, the variety of microorganisms is various, the climate is suitable for the growth of the microorganisms, the metabolic capability is strong, a large amount of ground source resources such as agricultural wastes can be converted into high-quality fermented feed in a short time, and therefore the production and development cost is reduced.
Limited conditions are few, Hainan has a natural environment which is unique, and the fermented feed is high in production efficiency. The production of the feed does not need to occupy a large space. A large amount of beneficial microorganisms, metabolites and digestive enzymes are generated in the fermentation process, which is beneficial to regulating the micro-ecological balance of intestinal tracts of organisms and improving the digestion, absorption and utilization of nutrient substances by animal organisms.
Fourthly, the local agricultural waste resources are effectively utilized, the waste and the environmental pollution are reduced, the production cost is reduced, and the ecological healthy feed has development potential and is popular with farmers at present.
Drawings
FIG. 1 shows a microscopic image (10X 100) of strain 3;
FIG. 2 shows a PCR map of strain 3; m: DL2000 DNA Marker; 3: strain 3;
FIG. 3 shows a phylogenetic tree of strain 3;
FIG. 4 shows a microscopic image (10X 100) of
FIG. 5 shows a PCR map of
FIG. 6 shows a phylogenetic tree of
FIG. 7 shows the response surface (7-1) and contour lines (7-2) of the interaction of the addition of the strain and the moisture content on the crude protein;
FIG. 8 shows the response surface (8-1) and contour lines (8-2) of the effect of strain addition and strain ratio interaction on crude protein;
FIG. 9 shows the response surface (9-1) and contour lines (9-2) for the effect of moisture content and strain ratio interaction on crude protein;
FIG. 10 shows the response surface (10-1) and contour lines (10-2) of the interaction of the amount of added bacteria and the moisture content on the influence of crude fat;
FIG. 11 shows the response surface (11-1) and contour line (11-2) of the effect of strain addition and strain ratio interaction on crude fat;
FIG. 12 shows the response surface (12-1) and contour lines (12-2) for the effect of moisture content and strain ratio interaction on crude fat;
FIG. 13 shows a process flow of desiccated coconut fermentation.
Detailed Description
The invention discloses a method for preparing biological feed by fermenting desiccated coconut with multiple strains, and a person skilled in the art can use the contents for reference and appropriately improve process parameters for realization. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
In the research, strains required by producing animal feed are separated and screened by taking naturally fermented desiccated coconut as a separation source, and molecular biology and physiological biochemical experiment identification are carried out to obtain microbial additive strains, namely saccharomyces cerevisiae and lactobacillus plantarum, which are allowed to be used by the national ministry of agriculture. The two-step fermentation is adopted, the desiccated coconut and the soybean meal are used as bottom materials, the saccharomyces cerevisiae, the bacillus subtilis and the lactobacillus plantarum are used as strains, the fermented feed is prepared through the two-step fermentation, the technological process and the fermentation conditions of the desiccated coconut fermented feed are optimized through a response surface method, the optimal fermentation process is obtained, and a foundation is laid for the development and utilization of the desiccated coconut in the future.
Screening of feeding fermentation microorganisms in desiccated coconut: separating, screening, purifying and performing physiological and biochemical identification on the fermented desiccated coconut by using an YPD culture medium and an MRS culture medium to obtain two strains of fermentation bacteria, which are respectively named as a strain 3 and a
The process for fermenting the desiccated coconut feed by the composite strains is optimized and researched by utilizing a response surface method, and the water content, the strain addition amount and the strain ratio are taken as main factors. Fermentation process parameters: the water content is 36%, 38% and 40%, the strain addition amount is 6%, 8% and 10%, and the strain ratio is yeast, bacillus subtilis and lactobacillus plantarum 54, 3.8, 36, 54, 5.7, 36, 27, 1.9 and 36, and the aerobic fermentation is carried out for 3 and 5 days, and the anaerobic fermentation is carried out for 5 and 15 days. The interaction effect between different conditions is taken into account. Selecting crude protein and crude fat as response values, and measuring the corresponding condition difference of maximization of different indexes. The optimal optimized fermentation conditions are selected to be aerobic fermentation for 3 days and anaerobic fermentation for 10 days, the strain addition amount is 10 percent, the water content is 36 percent, and the strain ratio is 54:3.8: 36. The content of crude protein and the content of crude fat in the fermented feed obtained under the condition are 35.25 percent and 31.56 percent respectively. The optimized desiccated coconut fermented feed obtained by the response surface model has accurate and reliable technological parameters, and has good practical production and application.
In the invention, the purchased saccharomyces cerevisiae, lactobacillus plantarum and bacillus subtilis are adopted to ferment the desiccated coconut according to the method, so that similar fermentation effects can be achieved.
The raw materials, strains, culture medium, reagents or instruments used in the method for preparing the biological feed by fermenting the desiccated coconut with the multiple strains can be purchased from the market.
The invention is further illustrated by the following examples:
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