CN111705015A - Microbial compound inoculant capable of inhibiting filamentous fungi and application thereof - Google Patents
Microbial compound inoculant capable of inhibiting filamentous fungi and application thereof Download PDFInfo
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- CN111705015A CN111705015A CN202010536538.XA CN202010536538A CN111705015A CN 111705015 A CN111705015 A CN 111705015A CN 202010536538 A CN202010536538 A CN 202010536538A CN 111705015 A CN111705015 A CN 111705015A
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- microbial
- fermentation
- bifidobacterium adolescentis
- fermented feed
- lactobacillus plantarum
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Abstract
The invention discloses a microbial compound inoculant capable of inhibiting filamentous fungi and application thereof, belonging to the technical field of biology and fermentation. The invention provides a microbial composite microbial inoculum with high safety and capable of effectively inhibiting filamentous fungi such as penicillium expansum, aspergillus niger, penicillium roqueforti, penicillium digitatum and the like, the microbial composite microbial inoculum consists of bifidobacterium adolescentis with the preservation number of GDMCC No.60925 and lactobacillus plantarum with the preservation number of CGMCC No.5494, and fermented feed prepared by the microbial composite microbial inoculum has rich nutrition, aromatic smell, difficult decay and high safety, and is specifically embodied as follows: the content of the mould in the fermented feed prepared by the microbial compound inoculant is 0CFU/g, and the content of the mould in the fermented feed prepared by the microbial compound inoculant is still 0CFU/g after the fermented feed is placed at 30 ℃ for 15 days.
Description
Technical Field
The invention relates to a microbial compound inoculant capable of inhibiting filamentous fungi and application thereof, belonging 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 microbial compound microbial inoculum which has high safety and can effectively inhibit filamentous fungi such as penicillium expansum, aspergillus niger, penicillium roqueforti, penicillium digitatum and the like.
[ solution ]
In order to solve the technical problems, the invention provides a microbial compound microbial inoculum, which comprises bifidobacterium adolescentis and lactobacillus plantarum;
the bifidobacterium adolescentis is preserved in the Guangdong province microbial strain preservation center with the preservation number of GDMCCNo.60925 and the preservation date of 2019, 12 months and 09 days;
the lactobacillus plantarum is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.5494, and the preservation date is 2011, 11 and 29 days.
In one embodiment of the invention, the viable bacteria ratio of the bifidobacterium adolescentis to the lactobacillus plantarum in the microbial composite inoculant is 1-2: 3-4.
In one embodiment of the invention, in the microbial composite bacterial agent, the ratio of viable bacteria of bifidobacterium adolescentis to viable bacteria of lactobacillus plantarum is 2: 3.
The invention also provides application of the microbial composite inoculant in inhibiting filamentous fungi, which is not aimed at diagnosis and treatment of diseases.
The invention also provides application of the microbial compound inoculant in preventing agricultural products or agricultural and sideline products from being rotted.
The invention also provides application of the microbial compound bacterium agent in preparation of fermented feed.
The invention also provides a method for preparing fermented feed by using the microbial compound inoculant, wherein the method comprises the steps of inoculating the microbial compound inoculant into fermentation raw materials for fermentation to obtain fermented feed; the fermentation feedstock comprises crop and/or crop waste.
In one embodiment of the present invention, the total viable count of bifidobacterium adolescentis and lactobacillus plantarum in the fermentation raw material is 1 × 109～1×1011CFU/g。
In one embodiment of the present invention, the total viable count of bifidobacterium adolescentis and lactobacillus plantarum in the fermentation raw material is 1.7 × 1010CFU/g。
In one embodiment of the invention, the fermentation feedstock contains water.
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 plant is a fruit, vegetable, oil and/or food crop.
In one embodiment of the invention, the crop waste is fruit waste, vegetable waste, oil crop waste and/or food 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 is to inoculate the microbial composite inoculant into a fermentation raw material consisting of apple pomace, bean pulp and water for fermentation to obtain the fermented feed.
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 ]
1. The invention provides a microbial composite microbial inoculum with high safety and capable of effectively inhibiting filamentous fungi such as penicillium expansum, aspergillus niger, penicillium roqueforti, penicillium digitatum and the like, the microbial composite microbial inoculum consists of bifidobacterium adolescentis with the preservation number of GDMCC No.60925 and lactobacillus plantarum with the preservation number of CGMCC No.5494, and fermented feed prepared by the microbial composite microbial inoculum has rich nutrition, aromatic smell, difficult decay and high safety, and is specifically embodied as follows:
(1) in the fermented feed prepared by the microbial compound inoculant, the content of crude protein is up to 24.45 percent, the content of crude fiber is as low as 27.34 percent, the content of crude fat is up to 10.72 percent, and the content of total amino acid is up to 7.34 percent;
(2) the content of the mould in the fermented feed prepared by the microbial compound inoculant is 0CFU/g, and the content of the mould in the fermented feed prepared by the microbial compound inoculant is still 0CFU/g after the fermented feed is placed at 30 ℃ for 15 days;
(3) the fermentation strains used by the microbial compound inoculant are bifidobacterium adolescentis and lactobacillus plantarum, which belong to probiotics, and are currently included in a strain list available for food issued by the ministry of health, so that potential safety hazards cannot be brought to human bodies.
2. The invention provides a method for preparing fermented feed, which comprises the following steps of inoculating a microbial compound microbial inoculum consisting of bifidobacterium adolescentis with the preservation number of GDMCCNo.60925 and lactobacillus plantarum with the preservation number of CGMCC No.5494 into a fermentation raw material containing crops and/or crop wastes for fermentation to obtain the fermented feed, wherein the fermented feed prepared by the method has rich nutrition, aromatic smell, difficult decay and high safety, and is specifically characterized in that:
(1) in the fermented feed prepared by the method, the content of crude protein is up to 24.45 percent, the content of crude fiber is as low as 27.34 percent, the content of crude fat is up to 10.72 percent, and the content of total amino acid is up to 7.34 percent;
(2) the content of the mould in the fermented feed prepared by the method is 0CFU/g, and after the fermented feed is placed at 30 ℃ for 15 days, the content of the mould in the fermented feed prepared by the method is still 0 CFU/g;
(3) the fermentation strains used by the method are bifidobacterium adolescentis and lactobacillus plantarum, and the bifidobacterium adolescentis and the lactobacillus plantarum belong to probiotics, are included in a strain list available for food issued by Ministry of health at present, and do not bring any potential safety hazard to human bodies.
Biological material preservation
The Bifidobacterium adolescentis (Bifidobacterium adolescentic) CCFM1108 is preserved in Guangdong province microbial strain preservation center in 12 months 09 in 2019, and the preservation number is GDMCCNo.60925, and the preservation address is No. 59 building and No.5 building of No. 100 college of Xielianlu Guangzhou city.
Drawings
FIG. 1: the effect of different concentrations of bifidobacterium adolescentis CCFM1108 fermentation supernatant on the growth of penicillium expansum mycelium.
FIG. 2: the effect of different concentrations of bifidobacterium adolescentis CCFM1108 fermentation supernatant on the amount of patulin synthesis.
FIG. 3: effect of Bifidobacterium adolescentis CCFM1108 fermentation supernatant on Penicillium expansum patA gene expression
FIG. 4: the influence of the fermented supernatant of bifidobacterium adolescentis CCFM1108 treated by different modes on the growth of the penicillium expansum mycelium.
FIG. 5: the effect of Lactobacillus plantarum CGMCC No.5494 on the growth of Penicillium expansum mycelium.
FIG. 6: the influence of the fermentation supernatant of lactobacillus plantarum CGMCC No.5494 with different concentrations on the permeability of mycelium cell membranes.
FIG. 7: the lactobacillus plantarum CGMCC No.5494 treated by different modes has the influence of fermentation supernatant 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; lactobacillus plantarum (Lactobacillus plantarum) CGMCC No.5494 (disclosed in the patent application publication No. CN 102586148A) mentioned in the following examples was deposited in the China general microbiological culture Collection center (CGMCC) without further patent program deposition; 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 lactobacillus content detection method comprises the following steps: the national standard GB/T4789.35-2016 lactic acid bacteria test 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 1-1: screening, identification, culture and observation of bifidobacterium adolescentis CCFM1108
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 (pH 6.5, 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 (pH 6.5, concentration 0.05M) containing 0.05% cysteine hydrochloride (M/M) and 0.25% Triton X-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 PCR reaction of the bacteria 16 SrDNA; extracting the genome of the strain obtained by screening in the step 1, amplifying and sequencing 18S rDNA of the strain (the nucleotide sequence of 16S rDNA obtained by amplification is shown as SEQ ID NO. 1), and comparing the obtained sequence with the nucleic acid sequence in GeneBank to obtain a strain which is bifidobacterium adolescentis and is named as bifidobacterium adolescentis CCFM 1108;
wherein, the PCR reaction system comprises: 10 × Taq buffer, 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 (3) selecting a single colony of the bifidobacterium adolescentis CCFM1108 screened in the step 1, inoculating the single colony on a mMRS solid culture medium, culturing for 48 hours at 37 ℃, and observing the colony characteristics of the bifidobacterium adolescentis CCFM1108 on the mMRS solid culture medium after 48 hours.
The observation shows that the colony diameter of the bifidobacterium adolescentis CCFM1108 on the mMRS solid culture medium ranges from 0.5 mm to 2mm, the front surface of the bifidobacterium adolescentis is circular, the side surface of the bifidobacterium adolescentis is in a protruded shape, the edge of the bifidobacterium adolescentis is neat, the bifidobacterium adolescentis is milky white or yellowish, the bifidobacterium adolescentis is opaque, the surface of the bifidobacterium adolescentis.
And (3) selecting a single colony of the bifidobacterium adolescentis CCFM1108 screened in the step 1, inoculating the single colony into a mMRS liquid culture medium, culturing at 37 ℃ for 48 hours, and observing the thallus characteristics of the bifidobacterium adolescentis CCFM1108 through an electron microscope after 48 hours.
It can be observed that bifidobacterium adolescentis CCFM1108 does not form spores and does not move, the thallus is rod-shaped and slightly bent, most of the thallus is V-shaped, and the minority of the thallus is Y-shaped, and the two ends are dark colored.
Selecting a single colony of the bifidobacterium adolescentis 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 OD600And (3) drawing a growth curve of the bifidobacterium adolescentis CCFM 1108.
As can be seen from the growth curve, the late logarithmic growth phase is reached when Bifidobacterium adolescentis CCFM1108 grows for 24 h.
Examples 1 to 2: effect of bifidobacterium adolescentis CCFM1108 and fermentation supernatant thereof on germination rate of filamentous fungus spores
1. Effect of Bifidobacterium adolescentis CCFM1108 on the germination rate of filamentous fungal spores (double-plate-growth inhibition method)
Selecting a single colony of the bifidobacterium adolescentis CCFM1108 obtained by screening in the embodiment 1, streaking the single colony on an mMRS solid culture medium, and culturing the single 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 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 to inoculate on a PDA inclined plane, culturing for 7d at 28 ℃, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; adding 5mL of sterile water into PDA culture medium with Penicillium expansum grown to the third generation, scraping spores with an inoculating loop, and filtering with 4 layers of sterile gauze to obtain Penicillium expansum spore suspensionDiluting Penicillium expansum spore suspension with sterile water to 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 medium4cfu/mL penicillium expansum spore suspension is cultured for 2d and 7d respectively at 28 ℃, an inhibition area (namely a streak area on the MRS solid culture medium) is observed, the percentage of the colony of the bifidobacterium adolescentis CCFM1108 in the inhibition area and the area without spore germination around the colony in the total area of the MRS solid culture medium is used as an index, the inhibition capacity of the bifidobacterium adolescentis CCFM1108 on the germination rate of penicillium expansum spores is detected, and the detection result is shown in Table 1.
The inhibition ability of bifidobacterium adolescentis CCFM1108 on the germination rates of spores of aspergillus niger, penicillium roqueforti and penicillium digitatum is respectively detected by referring to the same method, and the detection results are shown in Table 1.
As can be seen from table 1, when the inhibition ability of bifidobacterium adolescentis CCFM1108 on the germination rates of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum spores is detected, no spores germinate in the colony of bifidobacterium adolescentis CCFM1108 and the area not smaller than 70% of the mrs solid medium around the colony, and thus, the inhibition ability of bifidobacterium adolescentis CCFM1108 on the germination rates of penicillium expansum, aspergillus niger, penicillium roquefortii and penicillium digitatum spores is strong.
TABLE 1 inhibitory potency of Bifidobacterium adolescentis CCFM1108 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. Influence of Bifidobacterium adolescentis CCFM1108 fermentation supernatant on filamentous fungus spore germination rate (96-well plate-spore germination inhibition method)
Selecting a single colony of the bifidobacterium adolescentis CCFM1108 obtained by screening in the embodiment 1, streaking the single colony on an mMRS solid culture medium, and culturing the single 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 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。
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 fermentation supernatant of the bifidobacterium adolescentis CCFM1108 on the penicillium expansum, wherein the calculation result is shown in a 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 the bifidobacterium adolescentis CCFM1108 on aspergillus niger, penicillium roqueforti and penicillium digitatum is respectively detected by referring to the same method, and the detection result is shown in a table 2.
As can be seen from Table 2, the spore germination inhibition rates of the fermented supernatant of the Bifidobacterium adolescentis CCFM1108 on the penicillium expansum, the aspergillus niger, the penicillium roqueforti and the penicillium digitatum can be respectively as high as 99.88 +/-0.66%, 92.23 +/-0.53%, 88.83 +/-0.82% and 99.25 +/-0.62%, and therefore, the spore germination inhibition rates of the fermented supernatant of the Bifidobacterium adolescentis CCFM1108 on the penicillium expansum, the aspergillus niger, the penicillium roqueforti and the penicillium digitatum are stronger.
TABLE 2 inhibitory potency of Bifidobacterium adolescentis CCFM1108 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 |
Examples 1 to 3: effect of Bifidobacterium adolescentis CCFM1108 fermentation supernatant on growth of Penicillium expansum mycelium
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 added dropwise to the center of the plate, and incubated at 28 ℃ for 6 days, during which the effect of the fermentation supernatant of Bifidobacterium adolescentis CCFM1108 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 are shown in FIG. 1.
As shown in fig. 1, by measuring the diameter of the mycelium on the plate at the 6 th culture time, the inhibition capacity of the fermentation supernatant of bifidobacterium adolescentis CCFM1108 on the growth of penicillium expansum is increased along with the increase of the concentration of the fermentation supernatant, and the inhibition rate can reach 100% when the fermentation supernatant of bifidobacterium adolescentis 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.
Examples 1 to 4: effect of Bifidobacterium adolescentis CCFM1108 fermentation supernatant on 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 or 6d, adding 5mL of acidified water (pH 4.0) to the plate, standing for 1d or 1d, and scraping Penicillium expansum spores and mycelia on the plate; centrifuging and filtering 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 company) for quantification, and detecting the influence of fermented supernatant of Bifidobacterium adolescentis CCFM1108 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 figure 2; wherein, the conditions of the high performance liquid chromatography are as follows: color(s)Spectrum column: waters 4.6 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 inhibition ability of the fermentation supernatant of bifidobacterium adolescentis CCFM1108 on the yield of patulin produced by penicillium expansum is increased along with the increase of the concentration of the fermentation supernatant, and the inhibition rate can reach 83.5% when the fermentation supernatant of bifidobacterium adolescentis CCFM1108 reaches 20%; whereas an increase in the concentration of mrss liquid medium instead promotes patulin production.
Examples 1 to 5: influence of fermentation supernatant of bifidobacterium adolescentis CCFM1108 on expression level of penicillium expansum LaeA gene
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 flat plate respectively, dripping 10 muL of penicillium expansum spore suspension obtained in example 2 into the center of the flat plate, culturing at 28 ℃ for 6d and 6d, scraping penicillium expansum spores and mycelia on the flat plate, quickly freezing in 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 -tuqbin medium as internal reference to evaluate the influence of the bifidobacterium fermentation supernatant on the gene on the penicillium expansum gene synthesis (PatA) gene expression, wherein the relative quantity of the penicillium expansum gene is detected by adopting a secondary gene synthesis product expressed by RT-PCR (PatA) expression result, wherein the relative to detect the relative quantity of the penicillium expans-ΔΔCTThe method carries out quantitative calculation.
As shown in fig. 3, the fermentation supernatant of bifidobacterium adolescentis CCFM1108 can significantly reduce the relative expression of the LaeA gene of penicillium expansum; the mMRS liquid culture medium has no effect.
Examples 1 to 6: influence of temperature on the ability of bifidobacterium adolescentis CCFM1108 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 added dropwise to the center of the plate, and incubated at 28 ℃ for 6 days, during which time the effect of the Bifidobacterium adolescentis 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 are shown in FIG. 4.
As shown in FIG. 4, it was found that the inhibitory activity of the fermentation supernatant of Bifidobacterium adolescentis CCFM1108 (the diameter of mycelium growth: 8.8mm) on the growth of Penicillium expansum mycelium was slightly improved after the heat treatment as compared with the fermentation supernatant of Bifidobacterium adolescentis CCFM1108 (the diameter of mycelium growth: 9.0mm) without the heat treatment by measuring the diameter of mycelium on the plate at the 6 th day of culture.
Examples 1 to 7: influence of pH on the ability of Bifidobacterium adolescentis CCFM1108 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 incubated at 28 ℃ for 6 days during which the effect of pH on the ability of the fermentation supernatant of Bifidobacterium adolescentis CCFM1108 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 are shown in FIG. 4.
As shown in fig. 4, when the pH of the fermentation supernatant of bifidobacterium adolescentis CCFM1108 was 3.42 as measured by the diameter of the mycelium on the plate at the 6 th day of culture, the growth of penicillium expansum was strongly inhibited (the diameter of the mycelium was 9.0 mm); at pH 7, the inhibitory capacity of bifidobacterium adolescentis CCFM1108 fermentation supernatant on the growth of penicillium expansum mycelium was significantly reduced compared to that of bifidobacterium adolescentis CCFM1108 fermentation supernatant without pH adjustment (mycelium growth diameter was 15.6 mm).
Examples 1 to 8: effect of protease treatment on the ability of Bifidobacterium adolescentis CCFM1108 fermentation supernatant to inhibit the growth of Penicillium expansum mycelia
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 incubated at 28 ℃ for 6 days during which the effect of protease treatment on the ability of the fermentation supernatant of Bifidobacterium adolescentis CCFM1108 to inhibit 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 are shown in FIG. 4.
As shown in FIG. 4, the inhibitory activity of the supernatant from fermentation of Bifidobacterium adolescentis CCFM1108 (mycelium growth diameter: 8.6mm) on the growth of Penicillium expansum mycelium was slightly higher after the protease treatment than that of the supernatant from fermentation of Bifidobacterium adolescentis CCFM1108 (mycelium growth diameter: 9.0mm) without the protease treatment, as determined by measuring the diameter of mycelium on the plate at 6d of culture.
Example 2-1: lactobacillus plantarum CGMCC No.5494 and influence of fermentation supernatant thereof on germination rate of filamentous fungus spores
1. Influence of Lactobacillus plantarum CGMCC No.5494 on the germination rate of filamentous fungi spores (double-layer plate-growth inhibition method)
Dipping lactobacillus plantarum CGMCC No.5494 in a glycerin tube by using an inoculating loop, streaking on an MRS solid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a single colony; and selecting a single colony, inoculating the single colony in an MRS 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 MRS solid culture medium of bacterial suspension by using inoculating loop, culturing for 48 hours at 37 ℃, and adding 8mL of 1 × 10 with concentration on the MRS 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 an MRS solid culture medium) is observed, the percentage of the lactobacillus plantarum CGMCC No.5494 colony in the inhibition area and the area without spore germination around the colony to the total area of the MRS solid culture medium is used as an index, the inhibition capability of the lactobacillus plantarum CGMCC No.5494 on the germination rate of penicillium expansum spores is detected, and the detection result is shown in Table 3 and figure 5.
The inhibition ability of lactobacillus plantarum CGMCC No.5494 on the germination rates of spores of Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is respectively detected by referring to the same method, and the detection results are shown in Table 3.
As can be seen from Table 3 and FIG. 5, when the inhibition ability of Lactobacillus plantarum CGMCC No.5494 on the germination rate of spores of Penicillium expansum, Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is detected, 2d of cultivation is performed, the colonies of Lactobacillus plantarum CGMCC No.5494 in the inhibition area and the areas not smaller than 70% of the MRS solid medium around the colonies have no spore germination, 7d of cultivation is performed, and the colonies of Lactobacillus plantarum CGMCC No.5494 in the inhibition area and the areas not smaller than 30% of the MRS solid medium around the colonies have no spore germination. Therefore, the lactobacillus plantarum CGMCC No.5494 has stronger inhibition capability on the germination rate of spores of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum.
TABLE 3 inhibitory potency of Lactobacillus plantarum CGMCC No.5494 on spore germination rates of different filamentous fungi
Note: no spore germination exists in the lactobacillus plantarum colony and the area which is not less than 30% of the area of the plate around the colony +++; no spore germination in the lactobacillus plantarum colony area and the area around the colony which is less than 30% of the plate area ++; no spore germination in the colony area of lactobacillus plantarum only +; no zone of inhibition of spore germination-.
2. Influence of Lactobacillus plantarum CGMCC No.5494 fermentation supernatant on filamentous fungus spore germination rate (96-well plate-spore germination inhibition method)
Dipping lactobacillus plantarum CGMCC No.5494 in a glycerin tube by using an inoculating loop, scribing on an MRS solid culture medium, scribing on the MRS solid culture medium, and culturing for 48h at 37 ℃ in an anaerobic environment to obtain a single colony; selecting a single colony, inoculating the single colony in an MRS liquid culture medium, and culturing at 37 ℃ for 48h in an anaerobic environment to obtain a seed solution; inoculating the seed solution into an MRS 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 to inoculate on a PDA inclined plane, culturing for 7d at 28 ℃, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; in the growth ofAdding 5mL sterile water into PDA culture medium containing Penicillium expansum cultured to the third generation, scraping spores with inoculating loop, filtering with 4 layers of sterile gauze to obtain Penicillium expansum spore suspension, and 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 MRS liquid culture medium as control, and measuring OD of culture solution and MRS liquid culture medium580And calculating the spore germination inhibition rate of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant to the penicillium expansum, wherein the calculation result is shown in Table 2, wherein the spore germination inhibition rate (%) is × 100% of (1- (△ OD fermentation supernatant- △ ODMRS)/△ ODMRS).
The spore germination inhibition rates of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on Aspergillus niger, Penicillium roqueforti and Penicillium digitatum are respectively detected by referring to the same method, and the detection results are shown in Table 4.
As can be seen from Table 4, the spore germination inhibition rates of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum can be respectively as high as 99.68 +/-0.97%, 98.45 +/-0.13%, 96.35 +/-0.35% and 96.26 +/-0.54%, and thus the lactobacillus plantarum CGMCC No.5494 fermentation supernatant has stronger inhibition capability on the spore germination inhibition rates of the penicillium expansum, the aspergillus niger, the penicillium roquefortii and the penicillium digitatum.
TABLE 4 inhibitory potency of Lactobacillus plantarum CGMCC No.5494 on spore germination of different filamentous fungi
| Bacterial strains | Spore germination inhibition (%) |
| Penicillium expansum | 99.68±0.97 |
| Aspergillus niger | 98.45±0.13 |
| Blue mould of Mongolian blue | 96.35±0.35 |
| Penicillium digitatum | 96.26±0.54 |
Example 2-2: influence of lactobacillus plantarum CGMCC No.5494 fermentation supernatant on cell membrane permeability of expanded penicillium mycelium
Mixing MRS 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, 3.5:6.5, 4:6(MRS liquid culture medium: PDA culture medium) to obtain control group mixed solution with MRS liquid culture medium concentration of 10, 15, 20, 25, 30, 35, 45% (v/v); mixing fermentation supernatants of lactobacillus plantarum CGMCC No.5494 obtained in example 1 with PDA culture medium at volume ratio of 1:9, 1.5:8.5, 2:8, 2.5:7.5, 3:7, 3.5:6.5 and 4:6 respectively to obtain experimental group mixed liquid with fermentation supernatant concentration of 10, 15, 20, 25, 30, 35 and 45% (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 1d and 1d, scraping Penicillium expansum mycelium 1 on the plate, washing with PBS buffer solution twice, centrifuging at 8000rpm for 10min, and collecting mycelium 2; adding PI dye (purchased from Shanghai-constructed biological Co., Ltd.) with concentration of 1 μ M into the mycelium 2, incubating at room temperature (25 deg.C) for 30min, centrifuging at 8000rpm for 10min, and collecting mycelium 3; washing the mycelium 3 with PBS buffer solution twice, centrifuging at 8000rpm for 10min, and collecting mycelium 4; the staining of the mycelium 4 was observed with a fluorescence microscope and a picture was taken, which is shown in FIG. 6.
As shown in FIG. 6, the control group of mycelium 4 had no red fluorescence signal, indicating that the integrity of cell membrane of Penicillium expansum mycelium that was not treated with fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 was not destroyed; in the mycelium 4 of the experimental group, red fluorescence can be observed from the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 being 10%, but the fluorescence signal under the treatment is weaker, while a stronger fluorescence signal can be observed when the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is 25%, which shows that the cell membrane of the penicillium expansum can be damaged and the permeability of the penicillium expansum can be increased by the treatment of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494, and in addition, the concentration of the fermentation supernatant of the lactobacillus plantarum CGMCC No.5494 is in positive correlation with the change condition of the cell membrane permeability and the intensity of the fluorescence signal.
Examples 2 to 3: influence of Lactobacillus plantarum CGMCC No.5494 fermentation supernatant on growth of penicillium expansum mycelium
Mixing the MRS liquid culture medium with a PDA culture medium according to the volume ratio of 1.5:8.5(MRS liquid culture medium: PDA culture medium) to obtain a control group mixed solution with the concentration of the MRS liquid culture medium of 15 (v/v); mixing the fermentation supernatant obtained in example 1 with PDA medium at a volume ratio of 1.5:8.5 (fermentation supernatant: PDA medium) to obtain experimental group mixtures with respective fermentation supernatant concentrations of 15% (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, measuring the diameter of mycelium on each plate every 2d during the culturing period, and detecting the influence of fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of Penicillium expansum mycelium, with the detection result shown in FIG. 7; wherein the mycelium growth inhibition ratio (%) - (1- (D)Control group-DTreatment group) /DControl group) × 100%, wherein D is the diameter of mycelium.
As shown in FIG. 7, it can be seen from the measurement of the diameter of the mycelia on the plate at the 6 th day of culture that the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 can significantly inhibit the growth of the mycelia of Penicillium expansum with an inhibition rate as high as 35.8% (diameter of mycelia of 9.8 mm).
Examples 2 to 4: influence of temperature on ability of lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit growth of penicillium expansum mycelium
Mixing the fermentation supernatant obtained in example 1 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 1 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 added dropwise to the center of the plate and cultured at 28 ℃ for 6 days, during which the effect of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of the Penicillium expansum mycelia was examined by measuring the diameter of the mycelia on each plate every 2 days, and the results are shown in FIG. 7.
As shown in FIG. 7, it can be seen from the measurement of the diameter of the mycelia on the plate at the 6 th day of culture, the inhibitory activity of the fermentation supernatant (the diameter of the mycelia is 9.4mm) of Lactobacillus plantarum CGMCC No.5494 on the growth of the mycelia of Penicillium expansum after the heat treatment was slightly improved compared with the fermentation supernatant (the diameter of the mycelia is 9.8mm) of Lactobacillus plantarum CGMCC No.5494 without the heat treatment.
Examples 2 to 5: influence of pH on ability of lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit growth of penicillium expansum mycelium
Mixing the fermentation supernatant (pH 4) obtained in example 1 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 1 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 the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of mycelia on each plate every 2 days, and the results are shown in FIG. 7.
As shown in FIG. 7, the pH of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 was 4, which was measured by the diameter of mycelium on the plate at 6d, and the growth of Penicillium expansum was strongly inhibited (diameter of mycelium was 9.4 mm); when the pH value is 7, the inhibition capability (the diameter of the mycelium is 15.3mm) of the lactobacillus plantarum CGMCC No.5494 fermentation supernatant on the growth of the expanded penicillium mycelium is obviously reduced compared with the lactobacillus plantarum CGMCC No.5494 fermentation supernatant without pH adjustment.
Examples 2 to 6: influence of protease treatment on ability of lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit growth of penicillium expansum mycelium
Adjusting the pH of the fermentation supernatant obtained in example 1 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 1 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 Lactobacillus plantarum CGMCC No.5494 fermentation supernatant to inhibit the growth of Penicillium expansum mycelia was examined by measuring the diameter of mycelia on each plate every 2 days, and the results are shown in FIG. 7.
As shown in FIG. 7, by measuring the diameter of the mycelia on the plate at the 6d, the inhibitory activity of the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 on the growth of extended penicillium (diameter of mycelia: 9.7mm) was reduced after the treatment with protease compared to the fermentation supernatant of Lactobacillus plantarum CGMCC No.5494 without the treatment with protease (diameter of mycelia: 9.4 mm).
Example 3-1: 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 the bifidobacterium adolescentis CCFM1108 screened in the step 1 of the embodiment 1, inoculating the single colony into a mMRS liquid culture medium, and culturing at 37 ℃ for 48 hours to obtain a first-grade 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 tertiary seed liquid into a fermentation tank containing an MRS liquid culture medium in an inoculation amount of 2% (v/v), and performing amplification culture at 37 ℃ for 48h to obtain a fermentation liquid of the bifidobacterium adolescentis CCFM1108 (the whole fermentation liquid obtaining process needs to ensure aseptic operation, so that the mixed bacteria pollution is avoided);
(2) obtaining fermentation liquor of lactobacillus plantarum CGMCC No.5494 by referring to the method in the step (1);
(3) mixing the fermentation liquor of the bifidobacterium adolescentis CCFM1108 obtained in the step (1) with the fermentation liquor of the lactobacillus plantarum CGMCC No.5494 obtained in the step (2) to ensure that the ratio of the living bacteria of the bifidobacterium adolescentis to the living bacteria of the lactobacillus plantarum in the mixed bacteria liquid is 2:3, thus obtaining a microbial composite microbial inoculum;
(4) the method comprises the steps of controlling the particle sizes of apple pomace and soybean meal to be 250 microns (controlling the particle sizes through crushing and sieving), mixing the apple pomace and the soybean meal according to the mass ratio of 18:1, controlling the water content of a final fermentation raw material to be 60% through adding water, firstly filling the fermentation raw material into a fermentation bag (purchased from Wenzhou Xinghigao packaging Co., Ltd.) with a one-way exhaust valve, and then inoculating the fermentation liquid into the fermentation raw material in an inoculation amount of 1% (v/v), so that the total viable count of bifidobacterium adolescentis and lactobacillus plantarum in the fermentation raw material is 1.7 × 1010CFU/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 first scheme, the microbial compound inoculant is replaced by one or two of bifidobacterium adolescentis CCFM1108, lactobacillus plantarum CGMCC No.5494, lactobacillus plantarum GDMCC No.60604 (reference: Chenwei, Tianfengwei, Zhai and the like), lactobacillus plantarum capable of improving sleep and application thereof, namely, Chinese, 201910461904.7[ P ] 2019-08-19 or bifidobacterium adolescentis GDMCC No.60706 (reference: Chenwei, Wanggang, Qiqian and the like), bifidobacterium adolescentis CCFM1061 is applied to preparation of functional inocula, food and/or medicines, namely, Chinese, 201910765973.7[ P ] 2019-10-22 (see Table 5) respectively, and fermented feeds B-E are obtained.
The contents of crude protein, crude fiber, crude fat and total amino acids in the fermented feeds A to E were measured using the fermented raw materials after being left at 30 ℃ for 45 days as a blank control (see Table 6 for the results of the measurements).
The fermented raw materials after being placed at 30 ℃ for 3 days were used as blank controls, the pH values of the fermented feeds A to E were measured, and the contents of lactic acid and acetic acid in the fermented feeds A to E were measured (see Table 7 for the results of the measurements).
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-E (the detection result is shown in table 8).
As can be seen from Table 6, the fermented feed A contained crude protein as high as 24.45%, crude fiber as low as 27.34%, crude fat as high as 10.72%, and total amino acids as high as 7.34%.
As can be seen from Table 8, the mold content in the fermented feed A was 0CFU/g, and after standing at 30 ℃ for 15 days, the mold content in the fermented feed A was still 0CFU/g, and the effects of the fermented feeds B to E were much worse than those of the fermented feed A. The fermented feed A is not easy to breed filamentous fungi, not only is the lactic acid bacteria produce acid, but also the bifidobacterium adolescentis CCFM1108 and the lactobacillus plantarum CGMCC No.5494 can inhibit the filamentous fungi, and the effect of the double-bacteria fermentation of the bifidobacterium adolescentis CCFM1108 and the lactobacillus plantarum CGMCC No.5494 is better than that of the single-bacteria fermentation of the bifidobacterium adolescentis CCFM1108 or the lactobacillus plantarum CGMCC No. 5494.
TABLE 5 fermented feed A-E fermentation broth seed composition
| Group of | Bifidobacterium adolescentis | Lactobacillus plantarum |
| Fermented feed A | CCFM1108 | CGMCC No.5494 |
| Fermented feed B | CCFM1108 | - |
| Fermented feed C | - | CGMCC No.5494 |
| Fermented feed D | GDMCC No.60706 | CGMCC No.5494 |
| Fermented feed E | CCFM1108 | GDMCC No.60604 |
TABLE 6 content of crude protein, crude fiber, crude fat and total amino acids in fermented feeds A-E
| Group of | Crude protein/%) | Crude fiber/%) | Crude fat/%) | Total amino acid/%) |
| Blank control | 13.62 | 42.64 | 9.53 | 5.85 |
| Fermented feed A | 24.45 | 27.34 | 10.72 | 7.34 |
| Fermented feed B | 20.53 | 30.62 | 10.25 | 6.64 |
| Fermented feed C | 22.46 | 28.52 | 10.85 | 6.96 |
| Fermented feed D | 19.35 | 32.42 | 9.89 | 6.43 |
| Fermented feed E | 21.86 | 27.35 | 10.44 | 7.21 |
TABLE 7 pH values and lactic acid and acetic acid contents of fermented feeds A-E
| Group of | pH | Lactic acid (g/kg DM) | Acetic acid (g/kg DM) |
| Blank control | 4.21 | 0.23 | 0.42 |
| Fermented feed A | 3.48 | 1.31 | 0.73 |
| Fermented feed B | 3.98 | 1.28 | 0.82 |
| Fermented feed C | 3.85 | 1.23 | 0.47 |
| Fermented feed D | 3.62 | 1.19 | 0.66 |
| Fermented feed E | 3.54 | 0.92 | 0.59 |
TABLE 8 variation in the number of moulds in the fermented feeds A to E (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
<110> university of south of the Yangtze river
<120> a microbial complex inoculant capable of inhibiting filamentous fungi and application thereof
<160>3
<170>PatentIn version 3.3
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<211>1392
<212>DNA
<213> Bifidobacterium adolescentis
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gggcggtgtg tacaaggccc gggaacgcat tcaccgcggc gttgctgatc cgcgattact 120
agcgactccg ccttcatgga gtcgggttgc agactccaat ccgaactgag accggtttta 180
agggatccgc tccacctcgc ggtgtcgcat cccgttgtac cggccattgt agcatgcgtg 240
aagccctgga cgtaaggggc atgatgatct gacgtcatcc ccaccttcct ccgagttgac 300
cccggcggtc ccccgtgagt tcccaccacg acgtgctggc aacacagggc gagggttgcg 360
ctcgttgcgg gacttaaccc aacatctcac gacacgagct gacgacgacc atgcaccacc 420
tgtgaacccg ccccgaaggg agaccgtatc tctacggctg tcgggaacat gtcaagccca 480
ggtaaggttc ttcgcgttgc atcgaattaa tccgcatgct ccgccgcttg tgcgggcccc 540
cgtcaatttc tttgagtttt agccttgcgg ccgtactccc caggcgggat gcttaacgcg 600
ttggctccga cacggagacc gtggaatggt ccccacatcc agcatccacc gtttacggcg 660
tggactacca gggtatctaa tcctgttcgc tccccacgct ttcgctcctc agcgtcagtg 720
acggcccaga gacctgcctt cgccattggt gttcttcccg atatctacac attccaccgt 780
tacaccggga attccagtct cccctaccgc actcaagccc gcccgtaccc ggcgcggatc 840
caccgttaag cgatggactt tcacaccgga cgcgacgaac cgcctacgag ccctttacgc 900
ccaataattc cggataacgc ttgcacccta cgtattaccg cggctgctgg cacgtagtta 960
gccggtgctt attcgaaagg tacactcacc ccgaagggct tgctcccagt caaaagcggt 1020
ttacaacccg aaggccgtca tcccgcacgc ggcgtcgctg catcaggctt gcgcccattg 1080
tgcaatattc cccactgctg cctcccgtag gagtctgggc cgtatctcag tcccaatgtg 1140
gccggtcgcc ctctcaggcc ggctacccgt cgaagccatg gtgggccgtt accccgccat 1200
caagctgata ggacgcgacc ccatcccata ccgcaaaagc tttcccagag gaccatgcgg 1260
tcaactggag catccggcat taccacccgt ttccaggagc tattccggtg tatggggcag 1320
gtcggtcacg cattactcac ccgttcgcca ctctcaccca ggagcaagct cctgggatcc 1380
cgtcgactgc at 1392
<210>2
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<212>DNA
<213> Artificial sequence
<400>2
<210>3
<211>22
<212>DNA
<213> Artificial sequence
<400>3
tacggctacc ttgttacgac tt 22
Claims (10)
1. A microbial composite comprising Bifidobacterium adolescentis and Lactobacillus plantarum;
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 2019, 09 months 12;
the lactobacillus plantarum is preserved in China general microbiological culture Collection center (CGMCC), the preservation number is CGMCC No.5494, and the preservation date is 2011, 11 and 29 days.
2. The microbial composite bacterial agent according to claim 1, wherein the viable bacteria ratio of bifidobacterium adolescentis to lactobacillus plantarum in the microbial composite bacterial agent is 1-2: 3-4.
3. Use of the complex microbial inoculant according to claim 1 or 2 for inhibiting filamentous fungi without the purpose of diagnosis or treatment of diseases.
4. Use of the complex microbial inoculant according to claim 1 or 2 for preventing spoilage of agricultural or sideline products.
5. Use of the complex microbial inoculant of claim 1 or 2 in the preparation of fermented feed.
6. A method for preparing fermented feed by using a microbial compound inoculant, which is characterized in that the microbial compound inoculant of claim 1 or 2 is inoculated into a fermentation raw material for fermentation to obtain the fermented feed; the fermentation feedstock comprises crop and/or crop waste.
7. The method for preparing fermented feed using composite microbial inoculum according to claim 6, wherein the total viable count of Bifidobacterium adolescentis and Lactobacillus plantarum in the fermented raw material is 1 × 109～1×1011CFU/g。
8. The method for preparing fermented feed by using the microbial composite inoculant according to claim 6 or 7, wherein the crop wastes are fruit wastes, vegetable wastes, oil crop wastes and/or food crop wastes.
9. A fermented feed prepared by the method of any one of claims 6 to 8.
10. Use of the method of any one of claims 6 to 8 in the preparation of a fermented feed.
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| CN111705015B (en) | 2022-09-06 |
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