Feed containing pitaya stem protein, process for preparing feed by using microbiome solid state fermentation and application of feed

Abstract

The invention provides a pitaya stem protein-containing feed, a process for preparing the pitaya stem protein-containing feed through microbiome solid-state fermentation and application of the pitaya stem protein-containing feed, a main matrix of the pitaya stem protein-containing feed is pitaya stem powder obtained by drying and crushing pitaya stems, and the pitaya stems have relatively high nutritional values; the pitaya stems are mixed with other secondary matrixes to create conditions suitable for growth of strains, and then the strains are selected and reasonably matched, so that the nutrition of the fermented feed is further improved, the flavor of the traditional feed is improved, and the waste pitaya stems are recycled.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermented feed, and specifically relates to a pitaya stem protein-containing feed, a process for preparing the feed by solid-state fermentation of a microbial group, and an application thereof. Background Art

[0002] The feed formula structure of my country's animal husbandry industry is mainly composed of corn and soybean meal. Soybean meal is a byproduct obtained after soybeans are squeezed to produce edible oil. It is widely used in animal husbandry around the world because of its high protein content and amino acid composition similar to animal needs. my country's feed soybean meal mainly comes from imported soybeans, but the current international market soybean meal prices are rising rapidly and are unstable, which has brought certain troubles to the development of the domestic animal husbandry industry. The Ministry of Agriculture and Rural Affairs has promoted the reduction and substitution of feed soybean meal, and diversified formulas, less soybean meal, and reduced risks have become an industry consensus.

[0003] In addition, in modern farming, a small amount of functional additives are added to the feed while the animals are being fed, in order to promote their growth and prevent and treat diseases. Summary of the invention

[0004] The purpose of the present invention is to provide a protein feed containing pitaya stems, a process for preparing the same by solid-state fermentation of a microbiome and its application. The feed is produced using pitaya stem powder as the main raw material, thereby achieving rational utilization of resources and reducing waste, and also reducing the production proportion of soybean meal in the protein feed and reducing dependence on imported materials.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A process for preparing a feed containing pitaya stem protein by using microbial solid-state fermentation comprises the following steps:

[0007] (1) dispersing a fermentation substrate in water to obtain a fermentation substrate solution having a water-liquid-to-solid ratio of 1 to 1.5, wherein the fermentation substrate comprises a main substrate and a secondary substrate in a ratio of 40 to 60:49 to 80, the main substrate is pitaya stem powder, and the secondary substrate comprises at least one of soybean meal, corn flour, bran, wheat bran, fish meal and honey;

[0008] (2) inoculating a spore suspension of Aspergillus niger into a fermentation substrate, and then aerobically fermenting at 28 to 37° C. for 6 to 24 hours to obtain a first fermentation liquid, wherein the inoculation amount of the spore suspension of Aspergillus niger is 2 to 7 wt %;

[0009] (3) inoculating a microbial mixed bacterial liquid into the first fermentation liquid, and anaerobically fermenting at 28 to 37° C. for 5 to 14 days to obtain a pitaya stem protein feed, wherein the inoculation amount of the microbial mixed bacterial liquid is 5 to 15 wt %;

[0010] The mixed bacterial solution of the microbial group includes a seed bacterial solution of Lactobacillus casei, a seed bacterial solution of Saccharomyces cerevisiae and a seed bacterial solution of Bacillus subtilis in a volume ratio of 0.1-1:0.1-1:0.1-1. The bacterial solution concentration of the seed bacterial solution of Lactobacillus casei is 10 7 ~10 9 CFU / mL, the concentration of the seed culture of Saccharomyces cerevisiae is 10 7 ~10 9 The concentration of the seed culture of Bacillus subtilis was 10 7 ~10 9 CFU / mL.

[0011] Preferably, the mass ratio of pitaya stem powder, soybean meal, corn flour, bran, wheat bran, fish meal and honey is 40-60:20-25:10-20:10-15:5-10:2-5:2-5.

[0012] Preferably, the temperature of aerobic fermentation in step (2) is 33° C., and the time of aerobic fermentation is 12 h;

[0013] Preferably, the temperature of the anaerobic fermentation in step (3) is 33° C., and the time of the anaerobic fermentation is 10 days.

[0014] Preferably, the inoculation amount of the spore suspension of Aspergillus niger is 5wt%;

[0015] Preferably, the inoculation amount of the mixed bacterial solution of the microorganism group is 10wt%.

[0016] Preferably, the above process also includes step (4): drying the pitaya stem protein feed obtained in step (3) at 55-65°C, then crushing it to 40-80 mesh, then adding water to a moisture content of 20-40wt%, and finally preparing it into particles with a diameter of 3-15mm to obtain a granular pitaya stem protein feed.

[0017] Preferably, Lactobacillus casei is CGMCC1.8727;

[0018] Preferably, the Saccharomyces cerevisiae is CGMCC2.3973;

[0019] Preferably, the Bacillus subtilis spore strain is GCMCC1.821;

[0020] Preferably, the Aspergillus niger is CGMCC3.15663.

[0021] Preferably, the fermentation substrate is sieved with a 40-mesh screen after mixing.

[0022] Preferably, the water content of the pitaya stem powder is less than 10 wt %.

[0023] Further preferably, the pitaya stem powder is obtained by cutting, drying and crushing freshly pruned pitaya stems, the drying temperature is 55-65° C., and the crushed particle size is 20-60 mesh.

[0024] Preferably, the aqueous liquid-to-solid ratio of fermentation substrate to water is 1.25:1.

[0025] A pitaya stem protein-containing feed is prepared by the above process.

[0026] Application of the pitaya stem protein-containing feed in feeding animals.

[0027] The present invention has at least the following beneficial effects:

[0028] Firstly, the present invention uses pitaya stem powder as the main raw material to produce feed, thereby achieving rational use of resources and reducing waste, and also reducing the production proportion of soybean meal in protein feed and reducing dependence on imported materials.

[0029] Secondly, the raw material pitaya stem of the present invention is rich in nutrition, and is rich in flavonoids, terpenoid compounds, polysaccharides, vitamin E and other substances and niacin, calcium, phosphorus, iron and other components. By feeding the feed formula of the present invention, the nutritional intake of the raised animals is guaranteed, the immunity of the livestock is improved, and the disease resistance is enhanced.

[0030] Again, the present invention adopts multiple functional microorganisms to ferment the fermentation matrix containing pitaya stem powder, and by optimizing the fermentation conditions, without the need for complex pretreatment of the pitaya stem, solves the problems of insufficient nutrition and high crude fiber content in the feed containing pitaya stem powder, eliminates a large number of anti-nutritional factors, accumulates a large number of beneficial bacteria and metabolites, increases the protein content of the feed containing pitaya stem powder in the absence of an external nitrogen source, reduces the crude fiber content of the feed containing pitaya stem powder, retains the fragrant flavor of the pitaya stem powder, and improves the palatability of the feed.

[0031] Finally, pitaya stems have good antioxidant activity, which can play a certain protective role for finished feed products, extend the shelf life and reduce storage costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] The present invention will be further described below in conjunction with the accompanying drawings and embodiments, and the above advantages and features of the present invention will be more clearly stated.

[0033] Figure 1 is the standard curve of the number of live bacteria in the bacterial solution and the absorbance, where A is Bacillus subtilis; B is Saccharomyces cerevisiae; C is Lactobacillus casei;

[0034] Figure 2 The crude protein content results corresponding to the response surface design;

[0035] Figure 3 The crude fiber content results corresponding to the response surface design;

[0036] Figure 4 The effect of fermentation conditions on the crude protein content in fermented pitaya stem powder protein feed, where A is the relationship between temperature and fermentation time; B is the relationship between temperature and fermentation water-liquid ratio; C is the relationship between fermentation time and fermentation water-liquid ratio;

[0037] Figure 5 Figure 3 is the effect of fermentation conditions on the crude fiber content in fermented pitaya stem powder protein feed, where A is the relationship between temperature and fermentation time; B is the relationship between temperature and fermentation water-liquid ratio; C is the relationship between fermentation time and fermentation water-liquid ratio. DETAILED DESCRIPTION

[0038] The present invention is further described below in conjunction with the examples, but those skilled in the art should understand that the present invention is not limited to the following examples, and any improvements and changes made on the basis of the specific embodiments of the present invention are within the scope of protection of the claims of the present invention. The water-to-liquid ratio of the fermentation substrate solution (or the fermentation water-to-liquid ratio) mentioned in this application is the mass ratio of water to the fermentation substrate.

[0039] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0040] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0041] Lactobacillus casei (CGMCC1.8727), Saccharomyces cerevisiae (CGMCC2.3973), Bacillus subtilis (GCMCC1.821) and Aspergillus niger (CGMCC3.15663) described in the following examples are all purchased from China General Microbiological Culture Collection Center (CGMCC).

[0042] In the following embodiments, the PDA liquid medium has a formula of: 200 g potato, 20 g glucose, 20 g agar, 1000 mL distilled water, sterilized at 115° C. for 30 min; the MRS liquid medium has a formula of: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast powder, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g dipotassium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate, 0.04 g manganese sulfate, 1.0 g Tween-80, 15.0 g agar, 1000 mL distilled water, sterilized at 115°C for 30 min; the YPD liquid culture medium has a formula of: 10 g yeast extract, 20 g peptone, 20 g glucose, 20 g agar, 1000 mL distilled water, sterilized at 115°C for 30 min; the LB liquid culture medium has a formula of: 5 g yeast powder, 10 g tryptone, 10 g sodium chloride, 15 g agar, 1000 mL distilled water, sterilized at 121°C for 20 min.

[0043] [Example 1] Preparation of microbial strains and determination of bacterial solution concentration

[0044] 1. Activated strains:

[0045] Aspergillus niger can secrete enzymes related to lignin fiber, has the functions of degrading lignocellulose, inhibiting pathogens and producing flavor substances, Lactobacillus casei has the function of improving feed digestibility and palatability, Saccharomyces cerevisiae has the effects of providing high-quality feed protein, improving feed digestibility, inhibiting inflammatory response and maintaining intestinal homeostasis, and Bacillus subtilis has the function of improving immunity. Therefore, the present invention selects Aspergillus niger, Lactobacillus casei, Saccharomyces cerevisiae, and Bacillus subtilis as experimental strains. PDA, MRS, YPD, and LB liquid culture medium are configured, and the glycerol tubes taken out from the experimental strains in -80°C refrigerators are naturally thawed.

[0046] (1) inoculating Aspergillus niger spores into a PDA liquid culture medium, and culturing at 32° C. and 200 rpm for 3 days to obtain an Aspergillus niger seed culture solution;

[0047] (2) activating Lactobacillus casei and inoculating it into MRS liquid culture medium, and culturing it at 37° C. and 180 rpm for 12 h to obtain a Lactobacillus casei seed culture;

[0048] (3) activating the Saccharomyces cerevisiae and inoculating it into a YPD liquid culture medium, and culturing it at 30° C. and 200 rpm for 12 h to obtain a Saccharomyces cerevisiae seed culture solution;

[0049] (4) activating the Bacillus subtilis and inoculating it into LB liquid culture medium, and culturing it at 37° C. and 200 rpm for 12 h to obtain a Bacillus subtilis seed culture solution;

[0050] 2. Bacteria count

[0051] UV spectrophotometer (OD 600 ) was combined with plate counting to determine the concentration of Lactobacillus casei, Saccharomyces cerevisiae, and Bacillus subtilis. The original bacterial solutions of the three strains were diluted to a concentration gradient and plated. The plated bacterial solutions were counted by OD 600 Determination, using distilled water as blank control, plotting OD 600 The concentration of inoculated bacteria was 10 8 CFU / mL. The specific steps are as follows:

[0052] (1) Preparation of bacterial suspension: Under sterile operation, transfer 0.1 mL of the -80°C glycerol tube stock into 100 mL of liquid culture medium and culture at 180 rpm and 30°C for 10 h.

[0053] (2) Prepare 7 10 ml sterile centrifuge tubes in the clean bench, mark them with numbers, add 0.9 ml sterile water to each tube, take 0.1 ml of the initial bacterial solution into centrifuge tube No. 1, and so on, with a gradient of 10 from 10 to 10. -2 ~10 -7 Perform gradient dilution of bacterial solution. -4 , 10 -5 , 10 -6 , 10 -7 Take 0.1mL of each of the four bacterial solution gradients and spread it on a solid culture plate. After drying, invert it and culture it at 30℃ for 10h. Then use the plate counting method to calculate the number of colonies. When counting, select the plate with a colony count between 30 and 60. The bacterial solution concentration in the sample can be calculated according to its dilution multiple and sampling inoculum volume. Bacterial solution concentration = (colony number × dilution / 0.1CFU / mL).

[0054] (3) OD 600 The distilled water was used as blank control, the number of viable bacteria in the bacterial solution (CFU / mL) was used as the horizontal axis, and the OD of the bacterial solution was used as the horizontal axis. 600 The value is used as the vertical axis to draw the standard curve of the number of live bacteria in the bacterial solution and the absorbance. Figure 1 shown.

[0055] There is no need to count the strains of Aspergillus niger. When solid-state fermentation is carried out using pitaya stem powder as the base, the inoculation volume can be directly determined by the base volume.

[0056] [Example 2] Optimization of fermentation conditions of raw materials containing pitaya stem powder

[0057] Taking fermentation temperature (A), fermentation time (B) and fermentation water-liquid ratio (C) as factor variables, three levels of low, medium and high were selected for each factor, crude protein content was selected as the response value, and Design-Expert 10.0.8 was used to design a three-factor three-level experiment. The process steps are as follows:

[0058] (1) The pitaya stem is washed and dried at 60° C. to a water content of 10 wt %, and then crushed to 40 mesh to obtain pitaya stem powder.

[0059] (2) Pitaya stem powder, soybean meal, corn meal, bran and fish meal in a mass ratio of 50:25:10:10:5 were mixed and passed through a 40-mesh sieve as a fermentation matrix, and distilled water was added to the fermentation matrix according to the water-liquid-to-solid ratio (C) to obtain a fermentation matrix liquid.

[0060] (3) Inoculate 5 wt % of Aspergillus niger seed liquid and aerobically ferment at fermentation temperature (A) for 12 h to obtain a first fermentation liquid.

[0061] (4) inoculating the first fermentation liquid with a mixed bacterial solution of the microorganism group (including a seed bacterial solution of Lactobacillus casei, a seed bacterial solution of Saccharomyces cerevisiae, and a seed bacterial solution of Bacillus subtilis in a volume ratio of 1:1:1, and the bacterial solution concentration of each seed bacterial solution is 10 8 CFU / mL), the inoculation amount was controlled to be 10wt%, and anaerobically fermented at the fermentation temperature (A) for the fermentation time (B) to obtain a feed containing pitaya stem protein.

[0062] The response surface design and level table are shown in Table 1.

[0063] Table 1 Response surface design and level table

[0064]

[0065] According to the experimental group designed by response surface, a total of 17 groups of experiments were designed. To ensure the accuracy of the experiment, 3 groups of parallel control experiments were carried out at the same time. The response surface design scheme is shown in Table 2.

[0066] Table 2 Response surface design scheme

[0067]

[0068] Response surface analysis of crude protein content:

[0069] After fitting analysis by Design-Expert 10.0.8, the regression equation was obtained as follows: Crude protein content

[0070] =19.97+0.81×A+0.26×B-0.36×C+0.057×AB+0.28×AC+0.18×BC-1.29×A2 -0.79×B 2 -0.68×C 2 P<0.001, the model difference is significant.

[0071] Response surface analysis of crude fiber content:

[0072] After fitting analysis by Design-Expert 10.0.8, the regression equation was obtained as follows: Crude fiber content

[0073] =13.52-0.45×A-0.26×B+0.35×C-0.074×AB-0.067×AC+0.014×BC+1.23×A 2 +0.52×B 2 +0.82×C 2 P<0.001, the model difference is significant.

[0074] Figure 2 To design the response surface for the effect of the corresponding fermentation conditions on the crude protein content in the fermentation matrix containing pitaya stem powder; Figure 3 To design the response surface to determine the effect of fermentation conditions on the crude fiber content in the fermentation matrix containing pitaya stem powder; Figure 4 This is a schematic diagram of the effect of Design-Expert 10.0.8 fitting surface on crude protein content, where A is the relationship between the fermentation water-liquid-to-solid ratio and the fermentation temperature; B is the relationship between the fermentation water-liquid-to-solid ratio and the fermentation time; C is the relationship between the fermentation time and the fermentation temperature. Figure 5 The following is a schematic diagram of the effect of the Design-Expert 10.0.8 fitting surface on crude fiber content, where A is the relationship between the fermentation water-to-liquid ratio and the fermentation temperature; B is the relationship between the fermentation water-to-liquid ratio and the fermentation time; and C is the relationship between the fermentation time and the fermentation temperature. The above regression model was optimized using the response surface software, and it was found that the model was optimal when (A) the fermentation temperature was 33.45°C, (B) the fermentation time was 11.39d, and (C) the fermentation water-to-liquid ratio was 1.23, and the predicted crude protein content was 20.12wt%, and the predicted crude fiber content was 13.42wt%.

[0075] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A process for preparing a feed containing pitaya stem protein by solid-state fermentation of a microbial group, characterized in that: The steps include: (1) dispersing a fermentation substrate in water to obtain a fermentation substrate solution having a water-liquid-to-solid ratio of 1 to 1.5, wherein the fermentation substrate comprises a main substrate and a secondary substrate in a ratio of 40 to 60:49 to 80, the main substrate is pitaya stem powder, and the secondary substrate comprises at least one of soybean meal, corn flour, bran, wheat bran, fish meal and honey; (2) inoculating a spore suspension of Aspergillus niger into the fermentation substrate, and then aerobically fermenting at 28 to 37° C. for 6 to 24 hours to obtain a first fermentation liquid, wherein the inoculation amount of the spore suspension of Aspergillus niger is 2 to 7 wt %; (3) inoculating a microbial mixed bacterial liquid into the first fermentation liquid, and anaerobically fermenting at 28 to 37° C. for 5 to 14 days to obtain the pitaya stem protein feed, wherein the inoculation amount of the microbial mixed bacterial liquid is 5 to 15 wt %; The microbial group mixed bacterial liquid comprises a seed bacterial liquid of Lactobacillus casei, a seed bacterial liquid of Saccharomyces cerevisiae and a seed bacterial liquid of Bacillus subtilis in a volume ratio of 0.1-1:0.1-1:0.1-1, and the bacterial liquid concentration of the seed bacterial liquid of Lactobacillus casei is 10 7 ~10 9 CFU / mL, the bacterial solution concentration of the seed bacterial solution of Saccharomyces cerevisiae is 10 7 ~10 9 CFU / mL and the bacterial solution concentration of the seed bacterial solution of Bacillus subtilis is 10 7 ~10 9 CFU / mL.

2. The process according to claim 1, characterized in that The mass ratio of the pitaya stem powder, the soybean meal, the corn flour, the bran, the wheat bran, the fish meal and the honey is 40-60:20-25:10-20:10-15:5-10:2-5:2-5.

3. The process according to claim 1, characterized in that The temperature of the aerobic fermentation in step (2) is 33° C., and the time of the aerobic fermentation is 12 h; And / or, the temperature of the anaerobic fermentation in step (3) is 33° C., and the time of the anaerobic fermentation is 10 days.

4. The process according to claim 1, characterized in that The inoculation amount of the Aspergillus niger spore suspension is 5wt%; And / or, the inoculation amount of the mixed bacterial liquid of the microorganism group is 10wt%.

5. The process according to claim 1, characterized in that The method further comprises the step (4): drying the pitaya stem protein feed obtained in the step (3) at 55-65° C., crushing the feed to 40-80 mesh, adding water to a moisture content of 20-40 wt %, and finally preparing the feed to be granular with a diameter of 3-15 mm to obtain the pitaya stem protein feed.

6. The process according to claim 1, characterized in that The Lactobacillus casei is CGMCC1.8727; And / or, the Saccharomyces cerevisiae is CGMCC2.3973; And / or, the subtilis spore bacillus is GCMCC1.821; And / or, the Aspergillus niger is CGMCC3.15663.

7. The process according to claim 1, characterized in that The water content of the pitaya stem powder is less than 10 wt %.

8. The process according to claim 7, characterized in that The pitaya stem powder is obtained by cutting, drying and crushing freshly pruned pitaya stems, the drying temperature is 55-65° C., and the crushing particle size is 20-60 meshes.

9. A feed containing pitaya stem protein, characterized in that: The invention is prepared by the process described in any one of claims 1 to 8.

10. Use of the pitaya stem protein-containing feed according to claim 9 in feeding animals.

Images