CN111557377A - Method for preparing fruit and vegetable waste fermented feed - Google Patents
Method for preparing fruit and vegetable waste fermented feed Download PDFInfo
- Publication number
- CN111557377A CN111557377A CN202010398006.4A CN202010398006A CN111557377A CN 111557377 A CN111557377 A CN 111557377A CN 202010398006 A CN202010398006 A CN 202010398006A CN 111557377 A CN111557377 A CN 111557377A
- Authority
- CN
- China
- Prior art keywords
- fermented feed
- fermentation
- bifidobacterium
- bifidobacterium adolescentis
- pomace
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- 229940079839 sodium dehydroacetate Drugs 0.000 description 1
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Abstract
The invention discloses a method for preparing fruit and vegetable waste fermented feed, and belongs to the technical field of biology and fermentation. The invention provides a method for preparing fermented feed, which comprises the steps of inoculating Bifidobacterium adolescentis (Bifidobacterium adolescentic) with the preservation number of GDMCC No.60925 into a fermentation raw material containing crops and/or crop wastes for fermentation to obtain the fermented feed; the fermented feed prepared by the method has rich nutrition, aromatic smell,Is not easy to putrefy and has high safety, and the content of mould in the fermented feed prepared by the method is only 0.45 × 105CFU/g, and after being placed at 30 ℃ for 15 days, the content of the mould in the fermented feed prepared by the method is 0.
Description
Technical Field
The invention relates to a method for preparing fruit and vegetable waste fermented feed, and belongs to the technical field of biology.
Background
The apple is the first fruit in China, is one of the most favored crops, and has the advantages of wide planting range, large area and high yield. The apples in China are mostly used for producing the apple condensed juice, and a large amount of apple pomace can be discharged in the process of producing the apple condensed juice. According to statistics, in 2011, the yield of the apple pomace in China reaches 400 million tons. The effective utilization and treatment of the apple pomace become a big problem in the crop processing industry in China.
Besides apple pomace, the effective utilization and treatment problems of fruit wastes such as blueberry pomace remaining in blueberry juice production, mulberry pomace remaining in mulberry juice production, grape pomace remaining in grape juice production and the like, vegetable wastes such as hawthorn pomace remaining in hawthorn extract production, cassava pomace remaining in starch production, carrot pomace remaining in dried carrot production and the like, and oil crop wastes such as soybean meal remaining in soybean oil production, sesame meal remaining in sesame oil production and sunflower seed meal remaining in sunflower seed oil production are also a big problem in the crop processing industry in China.
Currently, there have been attempts to achieve effective utilization and disposal of crop wastes by preparing them into feeds. For example, Wuzhengke et al prepares the crop wastes into high protein fermented feed by mixed bacteria solid state fermentation (specifically, see references: Wuzhengke, Liu Guo Hua, Li Yang, etc.. the process optimization of the mixed bacteria solid state fermented rapeseed meal [ J ]. Chinese agricultural science, 2019,24: 4603-; a method for preparing fermented fruit residue feed from crop wastes by using an anaerobic fermentation method in a Hao forest and the like (see a reference document: the Hao forest, Zhouzing, Yuyuan good and the like; mulberry residue nutrient component analysis and fruit residue feed fermentation process research [ J ]. silkworm industry science, 2019,4: 563-one 568); plum-north et al prepared crop wastes into fermented feeds through lactobacillus fermentation (see specifically references: plum-north, plum permanence, yellow tolerance rise, etc.. manioc waste biofermentation feeds development design [ J ] light industry science and technology, 2019, 11: 30-31).
However, since agricultural wastes are rich in nutrients such as amino acids and water, and are easily attacked by filamentous fungi, filamentous fungi such as penicillium expansum, aspergillus niger, penicillium roqueforti, penicillium digitatum and the like (which are mainly responsible for crop spoilage by erosion of leaves, fruits and the like of crops and further cause storage diseases of crops, and which generate toxins such as patulin, citrinin and the like, which remain in crops and further enter human bodies through food chains and are potentially harmful) are one of the causes of crop spoilage, and therefore, even when sterilized, feeds prepared from crops as raw materials are easily spoiled during storage.
To solve the problem, the inventors of the Pinus massoniana and the like try to add a preservative into the feed prepared by using crops as raw materials to prolong the shelf life (the specific references can be found in Pinus massoniana and Wangwang. the influence of different mildewcides on the storage quality of the granulated feed [ J ]. the feed industry, 2019,40(9):38-44), but the addition of a large amount of the preservative has the problems of food safety, and the chemical additives have various limitations such as high cost, low return and adverse environmental protection (the specific references can be found in Yuzheng, Wuyingchao, Xijiayu and the like; the mildewcides dehydroacetic acid sodium dehydroacetate causes the rat bleeding test [ J ]. the animal medical progress, 2018,39(1):73-78 and the reference can be found in Kong Xueyang, Han Shumin, Lijinku and the like; the application of the mildewcides in the feed [ J ]. the feed science, 2019,40 (3-52), and insufficient addition of the preservative results in poor preservative effect.
Therefore, it is urgently required to find a method for preventing the deterioration of feed prepared from crops as raw materials with high safety and good effect.
Disclosure of Invention
[ problem ] to
The invention aims to provide a method for preventing feed prepared by using crops as raw materials from being rotten, which has high safety and good effect.
[ solution ]
In order to solve the technical problems, the invention provides a method for preparing fermented feed, which comprises the steps of inoculating Bifidobacterium adolescentis (Bifidobacterium adolescentic) into a fermentation raw material for fermentation to obtain the fermented feed; the fermentation feedstock comprises crops and/or crop waste; the bifidobacterium adolescentis is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No.60925 and the preservation date of 09 months at 2019.
In one embodiment of the present invention, the bifidobacterium adolescentis is inoculated into a fermentation raw material in the form of a seed liquid; the volume of the seed liquid accounts for 0.5-2% of the total volume of the fermentation raw materials.
In one embodiment of the present invention, the bifidobacterium adolescentis is inoculated into a fermentation raw material in the form of a seed liquid; the volume of the seed liquid accounts for 1 percent of the total volume of the fermentation raw materials.
In one embodiment of the invention, the water content of the fermentation raw material is 55-65%.
In one embodiment of the invention, the fermentation feedstock has a moisture content of 60%.
In one embodiment of the present invention, the fermentation temperature is 25-35 ℃ and the fermentation time is 2-5 days.
In one embodiment of the invention, the temperature of the fermentation is 30 ℃ and the time is 3 d.
In one embodiment of the invention, the fermentation process is kept anaerobic.
In one embodiment of the invention, the crop is a fruit, vegetable, oil and/or food crop; the crop waste is fruit waste, vegetable waste, oil crop waste and/or grain crop waste.
In one embodiment of the invention, the fruit waste is apple pomace, blueberry pomace, mulberry pomace and/or grape pomace.
In one embodiment of the invention, the vegetable waste is carrot pomace, cassava pomace and/or hawthorn pomace.
In one embodiment of the invention, the oil crop waste is soybean meal, cottonseed meal, peanut meal and/or rapeseed meal.
In one embodiment of the invention, the method comprises inoculating bifidobacterium adolescentis (bifidobacterium adolescentic) into a fermentation raw material for fermentation to obtain a fermented feed; the fermentation raw material consists of apple pomace, soybean meal and water.
In one embodiment of the invention, the mass ratio of the apple pomace to the soybean meal in the fermentation raw material is 16-20: 1-6.
In one embodiment of the invention, the mass ratio of the apple pomace to the soybean meal is 18: 1.
In one embodiment of the invention, the apple pomace has a particle size of 250 μm.
In one embodiment of the present invention, the particle size of the soybean meal is 250 μm.
The invention also provides the fermented feed prepared by the method.
The invention also provides application of the method in preparing fermented feed.
[ advantageous effects ]
The invention provides a method for preparing fermented feed, which comprises the steps of inoculating Bifidobacterium adolescentis (Bifidobacterium adolescentic) with the preservation number of GDMCCNo.60925 into a fermented raw material containing crops and/or crop wastes for fermentation to obtain the fermented feed; the fermented feed prepared by the method has rich nutrition, aromatic smell, difficult deterioration and high safety, and is specifically embodied in that:
(1) in the fermented feed prepared by the method, the content of crude protein is up to 20.53 percent, the content of crude fiber is as low as 30.62 percent, the content of crude fat is up to 10.25 percent, and the content of total amino acid is up to 6.64 percent;
(2) the content of mould in the fermented feed prepared by the method is only 0.45 × 105CFU/g, and after being placed for 15 days at the temperature of 30 ℃, the content of the mould in the fermented feed prepared by the method is 0;
(3) the fermentation strain used in the method is Bifidobacterium adolescentis (Bifidobacterium adolescentic), which is one of probiotics, and is currently included in a strain list available for food issued by the Ministry of health, so that no potential safety hazard is brought to human bodies.
Biological material preservation
A Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108, which is deposited in Guangdong province microbial strain collection center in 12-month 09 in 2019, wherein the deposit number is GDMCC No.60925, and the deposit address is No. 59 building 5 of Michelia Tokoro No. 100 Hospital, Guangzhou City.
Drawings
FIG. 1: effect of different concentrations of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatants on the growth of Penicillium expansum mycelium.
FIG. 2: the effect of different concentrations of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatants on the amount of patulin synthesis.
FIG. 3: effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant on Penicillium expansum patA Gene expression
FIG. 4: effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant treated in different ways on the growth of Penicillium expansum mycelium.
Detailed Description
The invention is further elucidated with reference to a specific embodiment and a drawing.
Penicillium expansum referred to in the following examples was purchased from China center for Industrial culture Collection of microorganisms with product number CICC 40658; the aspergillus niger related in the following examples is purchased from China center for culture Collection of industrial microorganisms, and the product number is CICC 2089; the Penicillium roqueforti related in the following examples is purchased from China center for culture Collection of industrial microorganisms with product number CICC 40663; penicillium digitatum referred to in the examples below was purchased from North Naita and has product number BNCC 336887; the apple pomace referred to in the following examples was purchased from Hengxing fruit juice Co., Ltd, Mei county, province, Shaanxi; the soybean meal referred to in the following examples was purchased from Hualong feed Co., Ltd, Fujian province.
The detection methods referred to in the following examples are as follows:
the water content detection method comprises the following steps: the sample is placed in an oven at 105 ℃ and dried to constant weight by adopting an oven drying method for determination, and the weight loss of the sample represents the moisture quality.
The crude protein detection method comprises the following steps: and (3) determining the content of crude protein in the sample by adopting a Kjeldahl method.
The crude fiber detection method comprises the following steps: the national standard GB/T6434-2006 'determination-filtration method for crude fiber in feed' is adopted.
The crude fat detection method comprises the following steps: the national standard GB/T6433-.
The total amino acid detection method comprises the following steps: the total amino acid content in the sample was determined by High Performance Liquid Chromatography (HPLC).
The pH detection method comprises the following steps: measured with a pH meter.
The organic acid detection method comprises the following steps: high performance liquid chromatography (reference: Xixia, screening of excellent lactic acid bacteria for silage and fermentation test research of apple pomace [ D ]. northwest agriculture and forestry science and technology university, 2014) is adopted.
The mould content detection method comprises the following steps: PDA plate dilution culture method (reference: Xixia, screening of excellent lactobacillus for silage and fermentation test research of apple pomace [ D ]. northwest agriculture and forestry science and technology university, 2014) is adopted.
The media involved in the following examples are as follows:
mrss solid medium: 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5 g of magnesium sulfate heptahydrate, 2g of diammonium citrate, 1g of cysteine hydrochloride and 18g of agar powder are added into 1L of distilled water, and the pH value is 6.2-6.5.
mrss liquid medium: adding 10g of peptone, 10g of beef extract, 20g of glucose, 5g of yeast extract, 2g of anhydrous sodium acetate, 0.25g of manganese sulfate monohydrate, 1mL of Tween 80, 2.6g of dipotassium phosphate trihydrate, 0.5 g of magnesium sulfate heptahydrate, 2g of diammonium citrate and 1g of cysteine hydrochloride into 1L of distilled water, wherein the pH value is 6.2-6.5.
PDA culture medium: 20g of glucose and 18g of agar were added to 1L of potato juice, and the pH was adjusted to the natural pH.
Example 1: screening, identification, culture and observation of Bifidobacterium adolescentis (CCFM 1108)
1. Screening
Taking 1g of a healthy adult excrement sample from a tin-free area, diluting the sample with physiological saline in a gradient manner, coating the diluted sample on an mMRS solid culture medium, culturing the sample in an anaerobic environment at 37 ℃ for 72 hours, and observing and recording colony morphology; selecting colonies, streaking on an mMRS solid culture medium, and performing purification culture at 37 ℃ in an anaerobic environment to obtain purified single colonies; selecting single colonies, streaking on mMRS solid culture medium, anaerobically culturing at 37 ℃ for 48h, performing gram staining on the obtained colonies (the gram staining method refers to textbook 'Industrial microbiology Breeding' author: Zhuge healthcare), recording the morphology of the colonies, examining the physiological and biochemical characteristics of the strains according to textbook 'common bacteria System identification Manual' (author: Dongxu pearl), and reserving the strains which are gram positive, smooth and round colonies and hydrogen peroxide negative, wherein the rest physiological and biochemical characteristics of the strains are as follows: can utilize D-ribose, L-arabinose, lactose, cellobiose, fructo-oligosaccharide, sorbitol, starch, glucose, mannose, xylose, maltose, trehalose; the nitrate reduction, catalase, arginine hydrolysis experiment and indole experiment are all negative.
2. Preliminary identification
Selecting a single colony of the strain obtained by screening in the step 1, inoculating the single colony into a mMRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 24 hours to obtain a bacterial liquid; centrifuging the bacterial liquid at 8000rpm for 2min, and collecting precipitate; washing the precipitate with phosphate buffer (pH6.5, concentration 0.05M) containing 0.05% cysteine hydrochloride (M/M) twice, centrifuging at 8000rpm for 2min, and collecting thallus; 0.2mg of the cells were resuspended in 200. mu.L of a phosphate buffer (pH6.5, concentration 0.05M) containing 0.05% cysteine hydrochloride (M/M) and 0.25% TritonX-100(M/M) to obtain a resuspension; adding 50 μ L of mixed solution (prepared by mixing sodium fluoride with concentration of 6mg/mL and sodium iodoacetate with concentration of 10 mg/mL) and 50 μ L of fructose-6-phosphate with concentration of 80mg/mL into the heavy suspension, and incubating at 37 deg.C for 1h to obtain incubation solution; adding 300 μ L of hydroxylamine hydrochloride (pH 6.5) with concentration of 0.139g/mL into the incubation solution, and standing at room temperature (25 deg.C) for 10min to obtain a solution to be detected; respectively adding 200 mu L of trichloroacetic acid solution with the concentration of 15% (M/M) and 200 mu L of HCL solution with the concentration of 4M into a solution to be detected to obtain a reaction system 1-2; 200 mu L of trichloroacetic acid solution with the concentration of 5% (M/M) and 200 mu L of HCL solution with the concentration of 0.1M are added into the reaction systems 1-2, and after the addition, the reaction systems 1-2 rapidly turn red, which indicates that the strain obtained by screening in the step 1 is positive in F6PPK and is primarily determined as bifidobacterium.
3. Further identification
Selecting a single colony of the strain obtained by screening in the step 1, inoculating the single colony into a mMRS liquid culture medium, and carrying out anaerobic culture at 37 ℃ for 24 hours to obtain a bacterial liquid; taking 1mL of the suspension to be placed in a 1.5mL centrifuge tube, centrifuging for 2min at 10000rpm, and collecting precipitates; washing the precipitate with sterile water once, centrifuging at 10000rpm for 2min, and collecting thallus; 0.2mg of the thallus is taken and resuspended in 500 mu L of sterile water for the bacteria 16SrDNAPCR reaction; extracting the genome of the strain obtained by screening in the step 1, amplifying and sequencing 18SrDNA of the strain (the nucleotide sequence of 16SrDNA obtained by amplification is shown as SEQ ID NO. 1), and comparing the obtained sequence with the nucleic acid sequence in GeneBank to show that the strain is bifidobacterium adolescentis and is named as bifidobacterium adolescentis (Bifidobacterium adolescentis) CCFM 1108;
wherein, the PCR reaction system comprises: 10 × Taqbuffer, 5 μ L; dNTP, 5. mu.L; 27F, 0.5 μ L; 1492R, 0.5 μ L; taq enzyme, 0.5. mu.L; template, 0.5 μ L; ddH2O, 38 μ L;
and (3) PCR reaction conditions: 95 ℃ for 5 min; 95 ℃ for 10 s; 30s at 55 ℃; 72 ℃ for 30 s; step2-4, 30 ×; 72 ℃ for 5 min; 2min at 12 ℃;
primers used for PCR: f: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO. 2); r: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO. 3).
4. Cultivation and Observation
And (2) selecting a single colony of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 screened in the step 1, inoculating the single colony on the mMRS solid culture medium, culturing at 37 ℃ for 48h, and observing the colony characteristics of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the mMRS solid culture medium after 48 h.
The observation shows that the colony diameter range of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the mMRS solid culture medium is 0.5-2 mm, the front form is circular, the side form is protruded, the edge is neat, the culture medium is milky white or light yellow, the culture medium is opaque, the surface is moist and smooth, and no pigment is generated.
And (2) selecting a single colony of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 obtained by screening in the step (1), inoculating the single colony into an mMRS liquid culture medium, culturing at 37 ℃ for 48h, and observing the thallus characteristics of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 through an electron microscope.
It can be observed that Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 does not form spores and does not move, and the cells are rod-shaped, slightly bent, mostly V-shaped, and rarely Y-shaped, and the two ends are dark colored.
Selecting a single colony of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 obtained by screening in the step 1, inoculating the single colony into a mMRS liquid culture medium, culturing at 37 ℃, and detecting the OD of the bacterial liquid every 4 hours in the culture process600By OD600The growth curve of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 was plotted.
From the growth curve, it was found that Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 reached the late logarithmic growth stage when it was grown for 24 hours.
Example 2: bifidobacterium adolescentis (CCFM 1108) and influence of fermentation supernatant thereof on germination rate of filamentous fungal spores
1. Effect of Bifidobacterium adolescentis (CCFM 1108) on the germination rate of filamentous fungal spores (double-layer plate-growth inhibition method)
Selecting a single colony of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 obtained by screening in example 1, streaking the single colony on an mMRS solid culture medium, and culturing the streaked colony for 48 hours at 37 ℃ in an anaerobic environment to obtain a single colony; and selecting a single colony, inoculating the single colony in an mMRS liquid culture medium, culturing for 48h at 37 ℃ in an anaerobic environment, and repeating the operation for 3 times to obtain a bacterial liquid cultured to the third generation.
Dipping the penicillium expansum bacterial liquid in an ampoule tube by using an inoculating loop, inoculating the penicillium expansum bacterial liquid on a PDA culture medium, culturing for 7d at 28 ℃ to obtain mycelium and spores, selecting the spores to inoculate on a PDA inclined plane, culturing for 7d at 28 ℃, repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation, adding 5mL of sterile water into the PDA culture medium in which the penicillium expansum cultured to the third generation grows, scraping the spores by using the inoculating loop, filtering by using 4 layers of sterile gauze to obtain penicillium expansum spore suspension, and diluting the penicillium expansum spore suspension by using the sterile water to the concentration of 1 × 104cfu/mL。
Dipping two parallel lines of two centimeters on the MRS solid culture medium of the bacterial suspension by using an inoculating loop, culturing for 48 hours at 37 ℃, and adding 8mL of 1 × 10 concentration on the mMRS solid culture medium4After cfu/mL penicillium expansum spore suspension is cultured for 2d and 7d respectively at 28 ℃, an inhibition area (namely a streak area on the mMRS solid culture medium) is observed, the percentage of a Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 colony in the inhibition area and a spore germination free area around the colony to the total area of the mMRS solid culture medium is used as an index, the inhibition capability of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the penicillium expansum spore germination rate is detected, and the detection result is shown in Table 1.
The inhibitory ability of Bifidobacterium adolescentis (CCFM 1108) to the spore germination rates of Aspergillus niger, Penicillium roqueforti and Penicillium digitatum was examined by the same method, and the results are shown in Table 1.
As can be seen from table 1, when the inhibitory activity of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the spore germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum is detected, no spore germinates exist in the colony of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 in the inhibitory region and in the region which is not less than 70% of the mrs solid medium around the colony, and thus the inhibitory activity of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the spore germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum is strong.
TABLE 1 inhibitory potency of Bifidobacterium adolescentis (CCFM 1108) on spore germination rates of different filamentous fungi
Note: no spore germination exists in the bifidobacterium colonies and the area which is not less than 30% of the area of the plate around the bifidobacterium colonies ++; no spore germination in the bifidobacterium colony area and the area around the colony which is less than 30 percent of the plate area ++; no spore germination in the colony area of bifidobacterium only +; no zone of inhibition of spore germination-.
2. Effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant on spore germination rate of filamentous fungi (96-well plate-spore germination inhibition method)
Selecting a single colony of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 obtained by screening in example 1, streaking the single colony on an mMRS solid culture medium, and culturing the streaked colony for 48 hours at 37 ℃ in an anaerobic environment to obtain a single colony; selecting a single colony, inoculating the single colony in an mMRS liquid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a seed solution; inoculating the seed solution into a mMRS liquid culture medium in an inoculation amount of 2% (v/v), culturing at 37 ℃ for 48h in an anaerobic environment, and repeating the operation for 2 times to obtain a fermentation liquid; the fermentation broth was centrifuged at 8000rpm for 10min and then filtered through a 0.2 μm filter to obtain a fermentation supernatant.
Dipping the penicillium expansum bacterial solution in the ampoule tube by using an inoculating ring, inoculating the penicillium expansum bacterial solution on a PDA culture medium, and culturing for 7d at 28 ℃ to obtain mycelium and spores;selecting spores, inoculating to PDA slant, culturing at 28 deg.C for 7d, repeating the operation for 2 times to obtain Penicillium expansum cultured to the third generation, adding 5mL sterile water into PDA culture medium containing Penicillium expansum cultured to the third generation, scraping spores with inoculating ring, filtering with 4 layers of sterile gauze to obtain Penicillium expansum spore suspension, diluting the Penicillium expansum spore suspension with sterile water to concentration of 1 × 104cfu/mL。
mu.L of fermentation supernatant was added to sterile 96-well plates and 10. mu.L of 1 × 104culturing cfu/mL penicillium expansum spore suspension at 28 ℃ for 48h to obtain a culture solution; using mMRS liquid culture medium as control, by measuring OD of culture solution and mMRS liquid culture medium580And calculating the spore germination inhibition rate of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant to penicillium expansum, wherein the calculation result is shown in table 2, wherein the spore germination inhibition rate (%) is × 100% of (1- (△ OD fermentation supernatant- △ ODmMRS)/△ ODmMRS).
The spore germination inhibition rate of the fermented supernatant of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on aspergillus niger, penicillium roqueforti and penicillium digitatum is respectively detected by the same method, and the detection result is shown in Table 2.
As can be seen from Table 2, the spore germination inhibition rates of the fermented supernatant of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 can reach 99.88 +/-0.66%, 92.23 +/-0.53%, 88.83 +/-0.82% and 99.25 +/-0.62% respectively, and the spore germination inhibition rates of the fermented supernatant of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 are relatively strong.
TABLE 2 inhibitory potency of Bifidobacterium adolescentis (CCFM 1108) on spore germination of different filamentous fungi
| Bacterial strains | Spore germination inhibition (%) |
| Penicillium expansum | 99.88±0.66 |
| Aspergillus niger | 92.23±0.53 |
| Blue mould of Mongolian blue | 88.83±0.82 |
| Penicillium digitatum | 99.25±0.62 |
Example 3: effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant on the growth of Penicillium expansum mycelia
Mixing mMRS liquid culture medium with PDA culture medium at volume ratio of 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7(mMRS liquid culture medium: PDA culture medium) to obtain control group mixed solution with mMRS liquid culture medium concentration of 10, 15, 20, 25, 30% (v/v); mixing the fermentation supernatants obtained in example 2 with PDA culture medium at volume ratios of 1:9, 1.5:8.5, 2:8, 2.5:7.5 and 3:7 (fermentation supernatant: PDA culture medium) to obtain experimental group mixed liquids with fermentation supernatant concentrations of 10, 15, 20, 25 and 30% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was dropped onto the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of Bifidobacterium adolescentis (CCFM 1108 fermentation supernatant on the growth of the mycelia of Penicillium expansum was examined by measuring the diameter of the mycelia on each plate every 2 days, and the results of the examination are shown in FIG. 1.
As shown in fig. 1, it can be seen from the measurement of the diameter of the mycelium on the plate at the 6 th day of culture that the inhibitory capacity of the fermentation supernatant of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the growth of penicillium expansum increases with the increase of the concentration of the fermentation supernatant, and the inhibitory rate can reach 100% when the fermentation supernatant of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 reaches 20%; while the change of the concentration of the mMRS liquid culture medium has no significant effect on the growth of the penicillium expansum.
Example 4: effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant on the yield of patulin produced by Penicillium expansum
Mixing mMRS liquid culture medium with PDA culture medium at volume ratio of 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7(mMRS liquid culture medium: PDA culture medium) to obtain control group mixed solution with mMRS liquid culture medium concentration of 10, 15, 20, 25, 30% (v/v); mixing the fermentation supernatants obtained in example 2 with PDA culture medium at volume ratios of 1:9, 1.5:8.5, 2:8, 2.5:7.5 and 3:7 (fermentation supernatant: PDA culture medium) to obtain experimental group mixed liquids with fermentation supernatant concentrations of 10, 15, 20, 25 and 30% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; dripping 10 μ L of Penicillium expansum spore suspension obtained in example 2 into the center of the plate, culturing at 28 deg.C for 6d and 6d, adding 5mL of acidified water (pH4.0) to the plate, standing for 1d and 1d, and scraping Penicillium expansum spores and mycelia on the plate; centrifuging spores and mycelia, collecting supernatant, filtering the supernatant with 0.22 μm filter membrane, loading by high performance liquid chromatography, comparing the result with commercial patulin standard (purchased from PULUBAN corporation) for quantification, and detecting the influence of fermented supernatant of Bifidobacterium adolescentis (CCFM 1108) on the yield of patulin produced by penicillium expansum according to the content of patulin in the supernatant, wherein the detection result is shown in FIG. 2; wherein, the conditions of the high performance liquid chromatography are as follows: a chromatographic column: waters4.6 x 250mm C18A chromatographic column; column temperature: 30 ℃; mobile phase: 10% acetonitrile, 90% water; flow rate: 1 mL/min; sample introduction amount: 10 mu L of the solution; a detector: UV; detection wavelength: 276 nm.
As shown in fig. 2, the inhibitory ability of the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant on the yield of patulin produced by penicillium expansum increases with the increase of the concentration of the fermentation supernatant, and the inhibitory rate can reach 83.5% when the Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant reaches 20%; whereas an increase in the concentration of mrss liquid medium instead promotes patulin production.
Example 5: effect of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant on expression level of LaeA gene of Penicillium expansum
Mixing mMRS liquid culture medium with PDA culture medium at a volume ratio of 1.5:8.5(mMRS liquid culture medium: PDA culture medium) to obtain control group mixed liquor with a mMRS liquid culture medium concentration of 15% (v/v), mixing fermentation supernatant obtained in example 2 with PDA culture medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA culture medium) to obtain experimental group mixed liquor with a fermentation supernatant concentration of 15% (v/v), pouring the mixed liquor of the control group and the experimental group into a plate, dripping 10 muL of Penicillium expansum spore suspension obtained in example 2 into the center of the plate, culturing at 28 ℃ for 6d and 6d, scraping Penicillium expansum spores and mycelia on the plate, quickly freezing the liquid nitrogen, storing at-80 ℃, grinding the Penicillium expansum spores and mycelia with liquid nitrogen, extracting total RNA by a Trizol method, performing RT-PCR by using β -tuqbulin medium as internal reference to evaluate the relative metabolite (metabolite) of Penicillium expansum gene expression on the supernatant, wherein the relative metabolite (metabolite) is detected by using a secondary strain FM 2, wherein the relative metabolite (metabolite of Penicillium expansum gene expression result is detected by using a strain FM 3-ΔΔCTThe method carries out quantitative calculation.
As shown in fig. 3, the relative expression level of the LaeA gene of penicillium expansum can be significantly reduced by fermenting the supernatant with Bifidobacterium adolescentis (CCFM 1108); the mMRS liquid culture medium has no effect.
Example 6: effect of temperature on the ability of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant to inhibit the growth of Penicillium expansum mycelium
Mixing the fermentation supernatant obtained in example 2 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); heating the fermentation supernatant obtained in example 2 at 121 ℃ for 20min, and mixing the fermentation supernatant with PDA culture medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA culture medium) to obtain an experimental group mixture with a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was dropped onto the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant on the growth of the Penicillium expansum mycelium was examined by measuring the diameter of the mycelium on each plate every 2 days, and the results of the examination are shown in FIG. 4.
As shown in FIG. 4, it was found that the inhibitory activity of the fermentation supernatant (mycelium growth diameter: 8.8mm) of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 on the growth of Penicillium expansum mycelium was slightly improved after the heat treatment as compared with the fermentation supernatant (mycelium growth diameter: 9.0mm) of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 without the heat treatment by measuring the diameter of the mycelium on the plate at the time of culturing at 6 d.
Example 7: effect of pH on the ability of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant to inhibit the growth of Penicillium expansum mycelium
Mixing the fermentation supernatant (pH 3.42) obtained in example 2 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); after adjusting the pH of the fermentation supernatant obtained in example 2 to 7 (adjusted with 1mol/L NaOH and 1mol/L HCl), the fermentation supernatant was mixed with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a mixture of experimental groups at a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was added dropwise to the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of pH on the ability of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of the mycelia on each plate every 2 days, and the results of the examination are shown in FIG. 4.
As shown in FIG. 4, when the pH of the fermentation supernatant of Bifidobacterium adolescentis (CCFM 1108) was 3.42, it was found that the growth of Penicillium expansum was strongly inhibited (the diameter of the grown mycelium was 9.0mm) by measuring the diameter of the mycelium on the plate at the 6 th day of culture; at pH 7, the inhibitory capacity of Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant on the growth of penicillium expansum was significantly reduced (mycelium growth diameter of 15.6mm) compared to Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 fermentation supernatant without pH adjustment.
Example 8: effect of protease treatment on the ability of Bifidobacterium adolescentis (CCFM 1108) fermentation supernatant to inhibit the growth of Penicillium expansum mycelium
Adjusting the pH of the fermentation supernatant obtained in example 2 to 7 (by using 1mol/L NaOH and 1mol/L HCl), adding 1mg/mL protease (purchased from SIGMA company, product number is P3910) into the fermentation supernatant, carrying out water bath at 37 ℃ for 2h, boiling the fermentation supernatant at 100 ℃ to inactivate the enzyme for 3min, and obtaining the fermentation supernatant treated by the protease; mixing the fermentation supernatant obtained in example 2 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain a control mixture with a fermentation supernatant concentration of 15% (v/v); mixing the fermentation supernatant with PDA culture medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA culture medium) to obtain an experimental group mixed solution with a fermentation supernatant concentration of 15% (v/v); pouring the mixed solution of the control group and the experimental group into a flat plate respectively; 10 μ L of the Penicillium expansum spore suspension obtained in example 2 was added dropwise to the center of the plate, and cultured at 28 ℃ for 6 days, during which the effect of protease treatment on the ability of Bifidobacterium adolescentis (CCFM 1108 fermentation supernatant to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of the mycelia on each plate every 2 days, and the results of the examination are shown in FIG. 4.
As shown in FIG. 4, by measuring the diameter of the mycelia on the plate at the 6 th day of culture, the inhibitory activity of the supernatant from fermentation of Bifidobacterium adolescentis (CCFM 1108) (growth diameter of mycelia was 8.6mm) against the growth of the mycelia of Penicillium expansum was slightly higher than that of the supernatant from fermentation of Bifidobacterium adolescentis (CCFM 1108) (growth diameter of mycelia was 9.0mm) without the protease treatment.
Example 9: preparation of fermented feed (apple pomace and bean pulp as raw materials)
The first scheme is as follows:
the method comprises the following specific steps:
(1) selecting a single colony of bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 obtained by screening in the step 1 of the embodiment 1, inoculating the single colony into a mMRS liquid culture medium, and culturing for 48 hours at 37 ℃ to obtain a primary seed solution; inoculating the primary seed liquid into an mMRS liquid culture medium in an inoculation amount of 2% (v/v), and culturing at 37 ℃ for 48h to obtain a secondary seed liquid; inoculating the secondary seed liquid into a seed tank containing mMRS liquid culture medium in an inoculation amount of 2% (v/v), and culturing at 37 ℃ for 48h to obtain a tertiary seed liquid; inoculating the three-stage seed liquid into a fermentation tank containing an mMRS liquid culture medium in an inoculation amount of 2% (v/v), and performing amplification culture at 37 ℃ for 48h to obtain a fermentation liquid (the whole fermentation liquid obtaining process needs to ensure sterile operation, so that the pollution of mixed bacteria is avoided);
(2) controlling the particle sizes of the apple pomace and the soybean meal to be 250 mu m (controlling the particle sizes by crushing and sieving), mixing the apple pomace and the soybean meal according to the mass ratio of 18:1, and adding water to control the water content of a final fermentation raw material to be 60%; the fermentation raw material is filled into a fermentation bag (purchased from Wenzhou Xingji high packaging Co., Ltd.) with a one-way exhaust valve, and then the fermentation liquid is inoculated into the fermentation raw material in the inoculation amount of 1% (v/v), so that the viable count of the bifidobacterium adolescentis CCFM1108 in the fermentation raw material is more than 109CFU/g, and then sealing the fermentation bag with the one-way exhaust valve to obtain a fermentation system; fermenting the fermentation system at 30 deg.C for 3d to obtain fermented feed A (apple pomace and soybean meal are subjected to high temperature instantaneous sterilization, and the whole fermented feed obtaining process needs to ensure aseptic operation to avoid mixed bacteria pollution).
Scheme II:
the method comprises the following specific steps:
on the basis of the scheme I, Bifidobacterium adolescentis (CCFM 1108) is replaced by Bifidobacterium adolescentis GDMCC No.60706 (reference: Chen Wei, Wang gang, Qin Qian and the like. application of Bifidobacterium adolescentis CCFM1061 in preparing functional microbial inoculum, food and/or medicament: China, 201910765973.7[ P ] 2019-10-22) or Bifidobacterium adolescentis GDMCC No.14395 (reference: Wang Yuan, Chen Wei, Wang gang and the like. Bifidobacterium adolescentis and application thereof: China, 201710963441.5[ P ] 2017-10-17) to obtain fermented feed B, C.
The contents of crude protein, crude fiber, crude fat and total amino acids in the fermented feeds A-C were determined using the fermented raw materials after being left at 30 ℃ for 3 days as a blank control (see Table 3 for the results of the determination).
The fermented raw materials after being placed at 30 ℃ for 3 days were used as blank controls to detect the pH values of the fermented feeds A to C and the contents of lactic acid and acetic acid in the fermented feeds A to C (see Table 4 for detection results).
And (3) taking the fermentation raw materials which are placed at 30 ℃ for 0-15 days as blank control, and detecting the number of the moulds in the fermentation feeds A-C (the detection result is shown in table 5).
As can be seen from Table 3, the fermented feed A contains crude protein up to 20.53%, crude fiber as low as 30.62%, crude fat as high as 10.25%, total amino acids as high as 6.64%, and is rich in nutrients.
As can be seen from Table 5, the content of mold in the fermented feed A was only 0.45 × 105CFU/g, and after being placed at 30 ℃ for 15 days, the content of the mould in the fermented feed prepared by the method is 0, but the fermented feed B-C has no effect. It is shown that the fermented feed A is less likely to grow filamentous fungi not only due to lactic acid bacteria producing acid but also due to Bifidobacterium adolescentis (CCFM 1108) itself inhibiting filamentous fungi.
TABLE 3 content of crude protein, crude fiber, crude fat and total amino acids in fermented feeds A-C
| Group of | Crude protein/%) | Crude fiber/%) | Crude fat/%) | Total amino acid/%) |
| Blank control | 13.62 | 42.64 | 9.53 | 5.85 |
| Fermented feed A | 20.53 | 30.62 | 10.25 | 6.64 |
| Fermented feed B | 19.23 | 35.82 | 9.78 | 5.99 |
| Fermented feed C | 16.34 | 33.46 | 9.67 | 6.36 |
TABLE 4 pH values and lactic acid and acetic acid contents of fermented feeds A-C
| Group of | pH | Lactic acid (g/kgDM) | Acetic acid (g/kgDM) |
| Blank control | 4.21 | 0.23 | 0.42 |
| Fermented feed A | 3.91 | 1.28 | 0.82 |
| Fermented feed B | 4.02 | 1.24 | 0.66 |
| Fermented feed C | 3.98 | 1.00 | 0.73 |
TABLE 5 variation in the number of moulds in fermented feeds A-C (10)5CFU/g)
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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Claims (10)
1. A method for preparing fermented feed is characterized in that Bifidobacterium adolescentis (Bifidobacterium adolescentic) is inoculated into a fermentation raw material for fermentation to obtain the fermented feed; the fermentation feedstock comprises crops and/or crop waste; the bifidobacterium adolescentis is preserved in Guangdong province microbial strain preservation center with the preservation number of GDMCC No.60925 and the preservation date of 09 months at 2019.
2. The method for preparing fermented feed according to claim 1, wherein the bifidobacterium adolescentis is inoculated into the fermentation raw material in the form of seed liquid; the volume of the seed liquid accounts for 0.5-2% of the total volume of the fermentation raw materials.
3. The method of claim 1 or 2, wherein the water content of the fermentation raw material is 55-65%.
4. A method of preparing a fermented feed according to any of claims 1 to 3, wherein the fermentation is carried out at a temperature of 25 to 35 ℃ for a period of 2 to 5 days.
5. A method of preparing a fermented feed according to any one of claims 1 to 4, wherein the crop is a fruit, vegetable, oil and/or food crop; the crop waste is fruit waste, vegetable waste, oil crop waste and/or grain crop waste.
6. The method of preparing fermented feed according to claim 5, wherein the fruit waste is apple pomace, blueberry pomace, mulberry pomace and/or grape pomace.
7. The method of preparing fermented feed according to claim 5, wherein the vegetable waste is carrot pomace, cassava pomace and/or hawthorn pomace.
8. The method of preparing fermented feed according to claim 5, wherein the oil crop waste is soybean meal, cottonseed meal, peanut meal and/or rapeseed meal.
9. A fermented feed prepared by the method of any one of claims 1 to 8.
10. Use of the method of any one of claims 1-8 for the preparation of fermented feed.
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| CN106306361A (en) * | 2016-08-25 | 2017-01-11 | 宁夏泰瑞制药股份有限公司 | Method for preparing biological fermentation feed |
| CN106333060A (en) * | 2016-08-26 | 2017-01-18 | 曹蕊 | Microbial fermentation feed and preparation method and application thereof |
| CN107446852A (en) * | 2017-08-28 | 2017-12-08 | 江南大学 | One lactobacillus plantarum and its application in terms of fermented feed |
| CN108634099A (en) * | 2018-04-26 | 2018-10-12 | 江苏惠田农业科技开发有限公司 | A kind of preparation method of blackberry pomace fermented feed |
| CN109497265A (en) * | 2018-12-17 | 2019-03-22 | 江苏春之雨生物科技发展有限公司 | It is a kind of using pomace, vinasse as the biological feedstuff mixed fungus fermentation method of raw material |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111705015A (en) * | 2020-06-12 | 2020-09-25 | 江南大学 | Microbial compound inoculant capable of inhibiting filamentous fungi and application thereof |
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