CN111206004B - Bifidobacterium longum and application thereof in inhibiting filamentous fungi - Google Patents
Bifidobacterium longum and application thereof in inhibiting filamentous fungi Download PDFInfo
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- CN111206004B CN111206004B CN202010161914.1A CN202010161914A CN111206004B CN 111206004 B CN111206004 B CN 111206004B CN 202010161914 A CN202010161914 A CN 202010161914A CN 111206004 B CN111206004 B CN 111206004B
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- bifidobacterium longum
- penicillium
- spores
- ccfm1109
- fermentation supernatant
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Abstract
The invention relates to bifidobacterium longum and application thereof in inhibiting filamentous fungi, belonging to the technical field of biology. The invention provides a bifidobacterium longum CCFM1109 strain, which can inhibit filamentous fungi and is specifically represented as follows: after the strain is respectively co-cultured with penicillium expansum spores, aspergillus niger spores, penicillium roqueforti spores or penicillium digitatum spores, the germination rate of the penicillium expansum, the aspergillus niger, the penicillium roqueforti or the penicillium digitatum spores can be obviously inhibited within 7 days; after the fermentation supernatant of the strain is co-cultured with penicillium expansum spores, aspergillus niger spores, penicillium roqueforti spores or penicillium digitatum spores respectively, the germination of the penicillium expansum spores, the aspergillus niger spores, the penicillium roqueforti spores or the penicillium digitatum spores can be obviously inhibited, and the spore germination inhibition rates can respectively reach 98.38 +/-0.37%, 90.23 +/-0.67%, 92.65 +/-1.27% and 96.34 +/-0.53%.
Description
Technical Field
The invention relates to bifidobacterium longum and application thereof in inhibiting filamentous fungi, belonging to the technical field of biology.
Background
Researches show that losses of different degrees exist in the whole supply chain process of fruits and vegetables from the initial planting to the final household consumption, and the losses are mainly caused by the problems of putrefaction, mechanical damage, fruit fly infection and the like of the fruits and vegetables in the processes of processing, transportation and storage after picking. Among them, spoilage is mainly caused by fungi, which causes appearance defects and nutritional deficiencies of fruits and vegetables. In developing countries with imperfect refrigeration and transportation facilities, the problem of fruit and vegetable spoilage is particularly prominent, and the loss of the fruit and vegetable industry is also particularly serious.
Penicillium expansum is a filamentous fungus belonging to the genus penicillium, which infects fruits and vegetables through mechanical damage suffered by the fruits and vegetables in the post-harvest processing process, and is one of the root causes of fruit and vegetable spoilage. Penicillium expansum can be isolated from fruit and vegetable surfaces in the United states, China, Japan, Italy, France, Iran, etc., and these isolated Penicillium expansum have been shown to produce toxins (mainly patulin and citrinin) that pose a significant threat to human health (see in particular references: Hammami W, Al Thani R, et Al. Patulin and patulin producing Penicillium sp. curing in applets and appl-based products including side food [ J ]. Journal of Infection in using furniture, 2017,11(4):343 349). In addition to fruit and vegetable surfaces, Penicillium expansum is also present inside the seeds of fruits, and under the appropriate conditions, its spores can penetrate through the hard seed coat to the soft vegetative tissue of the fruit, germinate and grow and produce toxins (mainly patulin and citrinin) (see in particular references S.Bandoh, M.Takeuchi, et al.Patulin distribution in degraded apple and its reduction [ J ]. International Biodegradation and Biodegradation,2009,63(4): 379-. In addition, studies have shown that besides the rotten tissue of fruits and vegetables, there are various toxin distributions in the good tissue around the rotten tissue of fruits and vegetables, and these toxins can spread even 10mm around the rotten tissue of fruits and vegetables, that is, even if the rotten part of fruits and vegetables is cut off, there are still a lot of residual toxins in the remaining intact part, and fruits and vegetables infected by Penicillium expansum can only be discarded completely, which further increases the loss of fruits and vegetables in the supply chain process (see in particular references: Fiorella Neri, Irene Donati, et al. evaluation of Penicillium expansum isolates for the growth of fruits and vegetables, growth and patulin accumulation in use and less common hosts,2010,143(3): 109-117). Therefore, inhibition of penicillium expansum is important to reduce losses in the fruit and vegetable industry.
Besides penicillium expansum, filamentous fungi such as aspergillus niger, penicillium roqueforti, penicillium digitatum and the like are also one of the original fierces of fruit and vegetable spoilage, which mainly erode leaves and fruits of fruits and vegetables to cause fruit and vegetable storage diseases, and can generate toxins, which remain in the fruits and vegetables to enter human bodies through food chains and have potential hazard. Therefore, it is also important to suppress filamentous fungi such as Aspergillus niger, Penicillium roqueforti, Penicillium digitatum and the like other than Penicillium expansum to reduce losses in the fruit and vegetable industry.
At present, fruit and vegetable spoilage caused by filamentous fungi is mainly controlled by chemical bactericides, but in view of the emergence of drug resistance of filamentous fungi and the potential health threat to human bodies caused by residual amounts of chemical bactericides, people tend to develop eco-friendly methods for controlling postharvest decay of fruits.
Compared with chemical bactericides, both physical control methods and biological control methods have certain advantages in the aspect of ecological friendliness. The physical prevention and control method is mainly used for inhibiting filamentous fungus infection by microwave heating and refrigeration of fruits and vegetables, but the physical prevention and control method has no continuity because the physical means such as microwave heating and refrigeration are short in stay on the surfaces of the fruits and vegetables.
The biological control method is mainly to inhibit filamentous fungi infection by antagonistic microorganisms, and the method is better in persistence compared with the physical control method, but the biological control method has the defect of poor effect, mainly because many antagonistic microorganisms belong to fungi and can cause fungal spoilage on the surface of fruits. Therefore, it is urgently needed to find a non-fungal antagonistic microorganism having a strong ability to inhibit filamentous fungi such as Penicillium expansum and the like so as to solve the defects of the existing biological control methods.
Disclosure of Invention
[ problem ] to
The technical problem to be solved by the invention is to provide a Bifidobacterium longum (Bifidobacterium longum) capable of inhibiting filamentous fungi.
[ solution ]
In order to solve the technical problems, the invention provides a Bifidobacterium longum (Bifidobacterium longum) CCFM1109, which is characterized in that the Bifidobacterium longum is preserved in Guangdong province microorganism strain preservation center with the preservation number of GDMCC No.60926 and the preservation date of 2019, 12 months and 09 days.
The Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is derived from healthy adult human fecal samples in Sichuan Chengdu areas, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1 through sequencing analysis, and the sequence obtained through sequencing is compared with the nucleic acid sequence in GeneBank, so that the result shows that the strain is the Bifidobacterium longum (Bifidobacterium longum) CCFM 1109.
The diameter range of the colony of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the mMRS solid culture medium is 0.5-2 mm, the front shape is circular, the side shape is in a protrusion shape, the edge is neat, the milk white is milky white and opaque, the surface is wet and smooth, and no pigment is generated.
The invention also provides a method for inhibiting filamentous fungi, which is characterized in that the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 and the filamentous fungi are co-cultured; or co-culturing the fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 and filamentous fungi; alternatively, the method comprises co-culturing the fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 and Bifidobacterium longum (Bifidobacterium longum) CCFM1109 with filamentous fungi. The fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is obtained by inoculating Bifidobacterium longum (Bifidobacterium longum) CCFM1109 into a culture medium for fermentation.
In one embodiment of the invention, inhibiting the filamentous fungus comprises reducing germination of spores of the filamentous fungus, inhibiting growth of mycelia of the filamentous fungus, reducing production of toxins by the filamentous fungus, reducing expression of a PatK gene in the filamentous fungus, reducing expression of a PatM gene in the filamentous fungus, and/or reducing expression of a PatN gene in the filamentous fungus.
The invention also provides a filamentous fungus inhibitor, and the components of the inhibitor comprise the Bifidobacterium longum (Bifidobacterium longum) CCFM 1109; or the inhibitor comprises fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM 1109; alternatively, the inhibitor comprises Bifidobacterium longum (Bifidobacterium longum) CCFM1109 and Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant. The fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is obtained by inoculating Bifidobacterium longum (Bifidobacterium longum) CCFM1109 into a culture medium for fermentation.
In one embodiment of the invention, inhibiting the filamentous fungus comprises reducing germination of spores of the filamentous fungus, inhibiting growth of mycelia of the filamentous fungus, reducing production of toxins by the filamentous fungus, reducing expression of a PatK gene in the filamentous fungus, reducing expression of a PatM gene in the filamentous fungus, and/or reducing expression of a PatN gene in the filamentous fungus.
In one embodiment of the invention, the components of the inhibitor further comprise a carrier.
In one embodiment of the invention, the carrier is a pharmaceutically acceptable carrier.
In one embodiment of the invention, the carrier is a filler, binder, wetting agent, disintegrant, lubricant and/or flavoring agent.
In one embodiment of the present invention, the inhibitor is in the form of powder, emulsion, granule or fine granule.
The invention also provides the application of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 or the method or the filamentous fungus inhibitor in inhibiting filamentous fungi, which is not aimed at diagnosing and treating diseases.
In one embodiment of the invention, the filamentous fungus comprises penicillium expansum, aspergillus niger, penicillium roqueforti and/or penicillium digitatum.
In one embodiment of the invention, inhibiting the filamentous fungus comprises reducing germination of spores of the filamentous fungus, inhibiting growth of mycelia of the filamentous fungus, reducing production of toxins by the filamentous fungus, reducing expression of a PatK gene in the filamentous fungus, reducing expression of a PatM gene in the filamentous fungus, and/or reducing expression of a PatN gene in the filamentous fungus.
The invention also provides the application of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 or the method or the filamentous fungus inhibitor in preventing fruit and vegetable spoilage.
[ advantageous effects ]
1. The invention provides a Bifidobacterium longum (Bifidobacterium longum) CCFM1109, which can inhibit filamentous fungi and is specifically embodied in that:
(1) after the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is co-cultured with penicillium expansum spores, aspergillus niger spores, penicillium roqueforti spores or penicillium digitatum spores respectively, the germination rate of the penicillium expansum, the aspergillus niger, the penicillium roqueforti or the penicillium digitatum spores can be obviously inhibited within 7 days;
(2) after the fermentation supernatant of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is co-cultured with penicillium expansum spores, aspergillus niger spores, penicillium roqueforti spores or penicillium digitatum spores respectively, the germination of the penicillium expansum spores, the aspergillus niger spores, the penicillium roqueforti spores or the penicillium digitatum spores can be obviously inhibited, and the spore germination inhibition rates can respectively reach 98.38 +/-0.37%, 90.23 +/-0.67%, 92.65 +/-1.27% and 96.34 +/-0.53%;
(3) after the penicillium expansum spores are inoculated to a culture medium containing the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant for culture, the growth of the penicillium expansum mycelium is obviously inhibited, and when the concentration of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant in the culture medium reaches 20% (v/v), the complete inhibition (namely the inhibition rate reaches 100%) can be achieved;
(4) after the penicillium expansum spores are inoculated to a culture medium containing the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant for culture, the yield of the penicillium expansum producing patulin is obviously inhibited, and when the concentration of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant in the culture medium reaches 20% (v/v), the complete inhibition (namely the inhibition rate reaches 100%) can be achieved;
(5) after the penicillium expansum spores are inoculated to a culture medium containing the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant for culture, the relative expression levels of the penicillium expansum PatK, PatM and PatN genes are obviously reduced to 0.11, 0.16 and 0.19 times of the penicillium expansum cultured by being inoculated to a culture medium without the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant respectively,
therefore, the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 has extremely high application prospect in inhibiting penicillium expansum and preventing fruit and vegetable spoilage.
2. The invention provides a Bifidobacterium longum (Bifidobacterium longum) CCFM1109, the fermentation supernatant of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 can inhibit penicillium expansum, and the fermentation supernatant of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 has good acid stability, thermal stability and protease stability, and the specific expression is that:
(1) the pH value of the fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is 3.56, and the inhibition capability of the fermentation supernatant on the growth of the expanded penicillium mycelium is stronger;
(2) after the heating treatment, the inhibition capability of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the growth of the penicillium expansum mycelium is not obviously changed compared with the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant which is not subjected to the heating treatment;
(3) after being treated by protease, the inhibiting ability of the fermentation supernatant of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the growth of the penicillium expansum mycelium is slightly improved compared with the fermentation supernatant of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 which is not treated by the protease,
therefore, the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 has extremely high application prospect in inhibiting filamentous fungi and preventing fruit and vegetable spoilage.
Biological material preservation
The Bifidobacterium longum (Bifidobacterium longum) CCFM1109 is preserved in Guangdong province microorganism strain preservation center in 2019 at 09 month 12, and the preservation number is GDMCC No.60926, and the preservation address is No. 59 building 5 of Michelia Tokoro No. 100 of Guangzhou city.
Drawings
FIG. 1: effect of different concentrations of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the growth of Penicillium expansum mycelium.
FIG. 2: effect of different concentrations of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the amount of patulin synthesis.
FIG. 3: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on penicillium expansum patA-F gene expression
FIG. 4: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 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 Nay and has the product number BNCC 336887.
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, 0.5g 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, 0.5g 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 longum
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 bacterial cells were resuspended in 500. mu.L of sterile water for bacterial 16S rDNAPCR reaction; 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 longum and is named as Bifidobacterium longum (Bifidobacterium longum) CCFM 1109;
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 (2) selecting a single colony of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 screened in the step (1) to be inoculated on a mMRS solid culture medium, culturing at 37 ℃ for 48h, and observing the colony characteristics of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the mMRS solid culture medium after 48 h.
The observation shows that the diameter range of the colony of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the mMRS solid culture medium is 0.5-2 mm, the front shape is circular, the side shape is convex, the edge is neat, the Bifidobacterium longum is milky white and opaque, the surface is moist and smooth, and no pigment is generated.
And (2) selecting a single colony of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 screened in the step (1) to be inoculated into an mMRS liquid culture medium, culturing at 37 ℃ for 48h, and observing the thallus characteristics of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 through an electron microscope after 48 h.
It can be observed that Bifidobacterium longum (Bifidobacterium longum) does not form spores and does not move, and the cells are rod-shaped, slightly curved, mostly V-shaped, and rarely Y-shaped, and have dark color at both ends.
Selecting the single colony of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 screened in the step 1, inoculating the single colony into an 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 longum (Bifidobacterium longum) CCFM1109 was plotted.
As can be seen from the growth curve, Bifidobacterium longum (Bifidobacterium longum) CCFM1109 reached the late logarithmic growth phase when grown for 24 hours.
Example 2: bifidobacterium longum (Bifidobacterium longum) CCFM1109 and influence of fermentation supernatant thereof on germination rate of filamentous fungus spores
1. Effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on spore germination rate of filamentous fungi (double-layer plate-growth inhibition method)
Selecting single colonies of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 obtained by screening in example 1, streaking the single colonies on an mMRS solid culture medium, and culturing the single colonies for 48 hours at 37 ℃ in an anaerobic environment to obtain single colonies; 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 liquid in the ampoule tube by an inoculating loop for inoculationCulturing on PDA culture medium at 28 deg.C and 200rpm for 7d to obtain mycelium and spore; selecting spores to inoculate on a PDA inclined plane, culturing at 28 ℃ and 200rpm for 7d, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; adding 5mL of sterile water into a PDA culture medium in which penicillium expansum grows and is cultured to the third generation, scraping spores by using an inoculating loop, and filtering by using 4 layers of sterile gauze to obtain penicillium expansum spore suspension; diluting 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 multiplied by 10 with the 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 MRS solid culture medium) is observed, the percentage of a Bifidobacterium longum (Bifidobacterium longum) CCFM1109 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 Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the germination rate of penicillium expansum spores is detected, and the detection result is shown in Table 1.
The inhibitory ability of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the germination rate of spores of Aspergillus niger, Penicillium roqueforti and Penicillium digitatum was measured by the same method, and the results are shown in Table 1.
As can be seen from table 1, when the inhibition ability of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the spore germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum is detected, no spore germinates exist in the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 colony in the inhibition area and the area which is not less than 70% of the mrs solid medium around the colony, and thus the inhibition ability of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the spore germination rate of penicillium expansum, aspergillus niger, penicillium roqueforti and penicillium digitatum is strong.
TABLE 1 inhibitory potency of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 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 longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on filamentous fungus spore germination rate (96-well plate-spore germination inhibition method)
Selecting single colonies of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 obtained by screening in example 1, streaking the single colonies on an mMRS solid culture medium, and culturing the single colonies for 48 hours at 37 ℃ in an anaerobic environment to obtain single colonies; 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 solution in the ampoule tube by using an inoculating ring, inoculating the penicillium expansum solution on a PDA culture medium, and culturing at 28 ℃ and 200rpm for 7d to obtain mycelia and spores; selecting spores to inoculate on a PDA inclined plane, culturing at 28 ℃ and 200rpm for 7d, and repeating the operation for 2 times to obtain penicillium expansum cultured to the third generation; adding 5mL of sterile water into a PDA culture medium in which penicillium expansum grows and is cultured to the third generation, scraping spores by using an inoculating loop, and filtering by using 4 layers of sterile gauze to obtain penicillium expansum spore suspension; diluting 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 1X 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 medium580Calculating the spore germination of Bifidobacterium longum (CCFM 1109) fermentation supernatant to Penicillium expansumThe inhibition rate and the calculation result are shown in table 2; wherein the spore germination inhibition ratio (%) is (1- (. DELTA.OD fermentation supernatant-. DELTA.ODmMRS)/. DELTA.ODmMRS) × 100%.
The spore germination inhibition rate of the fermented supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is respectively detected by the same method, and the detection results are shown in Table 2.
As can be seen from Table 2, the spore germination inhibition rates of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on Penicillium expansum, Aspergillus niger, Penicillium roqueforti and Penicillium digitatum can be respectively as high as 98.38 +/-0.37%, 90.23 +/-0.67%, 92.65 +/-1.27% and 96.34 +/-0.53%, and thus the inhibition capability of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the spore germination rates of Penicillium expansum, Aspergillus niger, Penicillium roqueforti and Penicillium digitatum is strong.
TABLE 2 inhibitory potency of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on spore germination of different filamentous fungi
| Bacterial strains | Spore germination inhibition (%) |
| Penicillium expansum | 98.38±0.37 |
| Aspergillus niger | 90.23±0.67 |
| Blue mould of Mongolian blue | 92.65±1.27 |
| Penicillium digitatum | 96.34±0.53 |
Example 3: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on growth of filamentous fungal 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 longum (Bifidobacterium longum) CCFM1109 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. 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 inhibition ability of the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant to the growth of the expanded penicillium mycelium increases with the increase of the concentration of the fermentation supernatant, and when the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant reaches 20%, complete inhibition (i.e. the inhibition rate reaches 100%) can be achieved; and the concentration change of the mMRS liquid culture medium has a promoting effect on the growth of penicillium expansum.
Example 4: effect of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the yield of patulin from 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); the fermentation supernatants obtained in example 2 were mixed at a ratio of 1:9, 1.5:8.5, 2:8,Mixing the culture medium PDA with the culture medium at a volume ratio of 2.5:7.5 and 3:7 (fermentation supernatant: PDA culture medium) to obtain experimental group mixed liquor with the concentrations of the fermentation supernatant 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 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 Pop), quantifying, and detecting the influence of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the yield of patulin produced by Penicillium expansum according to the patulin content 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: 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 Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant to the yield of patulin produced by penicillium expansum is increased along with the increase of the concentration of the fermentation supernatant, and complete inhibition (namely, the inhibition rate reaches 100%) can be achieved when the Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant reaches 20%; whereas an increase in the concentration of mrss liquid medium instead promotes patulin production.
Example 5: influence of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on PatA-F gene expression quantity 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 solution with mMRS liquid culture medium concentration of 15% (v/v); 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 mixture of experimental groups with a fermentation supernatant concentration of 15% (v/v); the mixed solution of the control group and the experimental group is poured respectivelyA flat plate; 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, scraping Penicillium expansum spores and mycelia on the plate, quick freezing with liquid nitrogen, and storing at-80 deg.C; grinding penicillium expansum spores and mycelium liquid nitrogen, extracting total RNA by adopting a Trizol method, and carrying out qRT-PCR by taking a beta-tubulin gene as an internal reference to evaluate the influence of fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 on the relative expression of patA-F genes (the PatA-F genes are genes for regulating and controlling synthesis of a secondary metabolite patulin by the penicillium expansum), wherein the detection result is shown in figure 3; wherein, the data adopts 2-ΔΔCTThe method carries out quantitative calculation.
As shown in FIG. 3, the relative expression level of PatK, PatM, and PatN genes in the fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 was significantly reduced, so that the relative expression levels of PatK, PatM, and PatN genes were reduced to 0.11, 0.16, and 0.19 times, respectively, that of Penicillium expansum cultured in a medium inoculated to a fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM 1109.
Example 6: effect of temperature on the ability of Bifidobacterium longum CCFM1109 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 longum (Bifidobacterium longum) CCFM1109 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: 5.3mm) of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 after heat treatment on the growth of the mycelia was not significantly changed from that of the fermentation supernatant (mycelium growth diameter: 5.7mm) of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 without heat treatment, by measuring the diameter of the mycelia on the plate at the 6 th day of culture.
Example 7: effect of pH on the ability of Bifidobacterium longum CCFM1109 fermentation supernatant to inhibit the growth of Penicillium expansum mycelium
Mixing the fermentation supernatant (pH 3.56) 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 longum (Bifidobacterium longum) CCFM1109 fermentation supernatant to inhibit the growth of 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 pH of the fermentation supernatant of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 was 3.56, and the growth of Penicillium expansum was strongly inhibited (the diameter of the grown mycelium was 5.7mm) by measuring the diameter of the mycelium on the plate at the 6 th culture day; at pH 7, the inhibitory activity of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant on the growth of Penicillium expansum was significantly reduced (mycelium growth diameter was 15.7mm) compared to Bifidobacterium longum (Bifidobacterium longum) CCFM1109 fermentation supernatant without pH adjustment.
Example 8: effect of protease treatment on the ability of Bifidobacterium longum CCFM1109 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 longum (Bifidobacterium longum) CCFM1109 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, it was found that the inhibitory activity of the supernatant from the fermentation of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 (mycelium growth diameter: 5.0mm) against the growth of the mycelia was slightly higher after the protease treatment than that of the supernatant from the fermentation of Bifidobacterium longum (Bifidobacterium longum) CCFM1109 (mycelium growth diameter: 5.7mm) without the protease treatment, as measured by the diameter of the mycelium on the plate at the 6 th day of the culture.
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> Bifidobacterium longum and application thereof in inhibiting filamentous fungi
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<170> PatentIn version 3.3
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tacaccggga attccagtct cccctaccgc actcaagccc gcccgtaccc ggcgcggatc 840
caccgttaag cgatggactt tcacaccgga cgcgacgaac cgcctacgag ccctttacgc 900
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Claims (9)
1. Bifidobacterium longum (Bifidobacterium longum) is preserved in Guangdong province microorganism culture collection with the preservation number of GDMCC No.60926 and the preservation date of 2019, 12 months and 09 days.
2. A method for inhibiting filamentous fungi, which comprises co-culturing the Bifidobacterium longum of claim 1 with filamentous fungi; alternatively, the method comprises co-culturing Bifidobacterium longum according to claim 1, a fermentation supernatant of Bifidobacterium longum according to claim 1, and a filamentous fungus; the filamentous fungi are Penicillium expansum, Aspergillus niger, Penicillium roqueforti and/or Penicillium digitatum, and the methods are not directed to the diagnosis and treatment of disease.
3. A filamentous fungus inhibitor, wherein the inhibitor comprises the bifidobacterium longum of claim 1; alternatively, the inhibitor component comprises bifidobacterium longum according to claim 1 and a fermentation supernatant of bifidobacterium longum according to claim 1.
4. The inhibitor of claim 3, wherein the inhibitor component further comprises a carrier.
5. The filamentous fungus inhibitor according to claim 4, wherein said carrier is a pharmaceutically acceptable carrier.
6. A filamentous fungus inhibitor according to claim 3 or 4, wherein the carrier is a filler, a binder, a wetting agent, a disintegrant, a lubricant and/or a flavouring agent.
7. The filamentous fungus inhibitor according to claim 6, wherein the inhibitor is in the form of powder, emulsion, granule or fine granule.
8. Use of bifidobacterium longum according to claim 1, or of a filamentous fungus inhibitor according to any of claims 3 to 7 for inhibiting filamentous fungi not aimed at the diagnosis or treatment of diseases, characterized in that the filamentous fungi are penicillium expansum, aspergillus niger, penicillium roqueforti and/or penicillium digitatum.
9. Use of a bifidobacterium longum according to claim 1, or a method according to claim 2, or a filamentous fungus inhibitor according to any of claims 3 to 7 for preventing spoilage of fruits and vegetables, wherein the spoilage of fruits and vegetables is caused by filamentous fungi selected from the group consisting of penicillium expansum, aspergillus niger, penicillium roqueforti and/or penicillium digitatum.
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