Method and kit for screening edible and medicinal fungi with high yield of microbial proteins and target active components

Abstract

The invention discloses a method for screening edible and medicinal fungi with high yield of microbial proteins and target active components and a kit thereof, and belongs to the technical field of microbial screening. The method comprises the following steps: carrying out primary culture screening on a culture medium containing a specific precursor; performing safe primary screening; carrying out miniaturized culture and rapid protein detection on the safe strains to evaluate the protein potential, and meanwhile, carrying out visual activity screening by utilizing a report system based on functional characteristics of target active ingredients; and finally, carrying out performance verification on the optimized strain. According to the method disclosed by the invention, high-throughput and synchronous evaluation of the protein yield and the active component synthesis potential of the strain is realized, the bottlenecks of low throughput and single target of a traditional method are solved, the excellent edible and medicinal fungus strain with double effects of one strain can be quickly screened, and a core strain screening tool is provided for efficient production of sustainable protein and natural active components.

Description

Technical Field

[0001] This invention belongs to the field of microbial screening technology, specifically relating to a method and kit for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients. Background Technology

[0002] With global population growth, the demand for sustainable protein is becoming increasingly urgent. Traditional livestock farming for animal protein production suffers from unsustainable problems such as high resource consumption and a large environmental footprint. The fermentation of edible and medicinal fungi (such as the mycelium of various mushrooms) to produce microbial protein offers significant advantages, including extremely high land use efficiency, rapid growth, no climate limitations, and the ability to be vertically produced, making it one of the key potential solutions to address future protein supply gaps.

[0003] Meanwhile, edible and medicinal fungi (such as Ganoderma lucidum, Cordyceps, Poria cocos, and Phellinus linteus) are recognized as a treasure trove of natural active ingredients. The polysaccharides, terpenes, sterols, and polyphenols produced by their metabolism possess various biological activities, including immunomodulation, anti-tumor activity, antioxidant activity, and lipid-lowering activity, and are in huge demand in the health industry. Integrating the production of high-nutritional-value proteins with the production of high-value-added active ingredients into the same fermentation process can greatly improve resource utilization efficiency and product economic value, representing an important direction for industrial upgrading.

[0004] However, traditional methods for screening edible and medicinal fungi have significant limitations: they primarily rely on plate culture and shake-flask fermentation, resulting in low throughput and long cycles, making it difficult to rapidly identify target strains from vast microbial resource banks. More importantly, traditional methods typically screen only for a single trait, such as measuring only biomass or crude protein content, or detecting only a specific active ingredient, failing to simultaneously assess the strain's comprehensive potential in both protein production and active ingredient synthesis. This leads to screened strains often having limited functions, failing to meet the demand for strains with "all-around utilization," and hindering the development of related industries.

[0005] Therefore, there is an urgent need in this field for an integrated screening method that can perform high-throughput, rapid, and simultaneous evaluation of protein yield and target active ingredient synthesis capabilities of edible and medicinal fungal strains. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method and kit for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients.

[0007] To solve the above-mentioned technical problems, the present invention provides the following technical solution:

[0008] The first objective of this invention is to provide a method for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients, comprising the following steps:

[0009] Step (1): Primary screening: The edible and medicinal fungal strains to be tested are inoculated on the primary screening medium and cultured. Preliminary screening is carried out based on the colony growth rate and morphological uniformity.

[0010] Step (2): Safety screening: For the strains obtained from the initial screening, safety assessment is performed based on colony morphology characteristics and / or molecular toxicity markers to eliminate potentially unsafe strains;

[0011] Step (3): Re-evaluation and screening: For strains that pass the initial safety screening, the following two types of evaluations are performed in parallel or sequentially:

[0012] a) Assess protein yield potential using miniaturized culture and rapid protein detection methods;

[0013] b) Visual activity screening using a reporting system based on the functional properties of the target active ingredient;

[0014] Step (4): Performance verification and evaluation: Fermentation verification and product evaluation are carried out on the candidate strains obtained from the re-evaluation.

[0015] Preferably, in step (1), the edible and medicinal fungal strain is at least one of the following: Ganoderma, Poria, Cordyceps, Phellinus, Hericium, and Lentinus.

[0016] The primary screening culture medium contains specific precursor substances that are beneficial to the biosynthesis of the target active ingredient.

[0017] Preferably, the target active ingredient is at least one of polysaccharides, terpenoids, polyphenols, sterols, and antibacterial proteins.

[0018] The specific precursor substances include at least one of lanosterol, phenylalanine, coumaric acid, mevalonic acid, sucrose, and amino acids.

[0019] Preferably, in step (1), the primary screening culture medium contains one or more non-synthetic nitrogen sources.

[0020] Preferably, the non-synthetic nitrogen source includes soybean meal, cottonseed meal, corn steep liquor powder, yeast extract, or insect peptone.

[0021] Preferably, in step (2), the safety assessment of colony morphology characteristics includes whether abnormal pigments are produced and whether the hyphal morphology is normal; the safety assessment using molecular toxicity markers includes molecular marker detection based on toxin-related genes.

[0022] Preferably, in step (3)a), assessing protein yield potential using miniaturized culture and rapid protein detection methods includes the following steps:

[0023] The strain was inoculated into a miniaturized culture system for liquid fermentation, and the protein content of the fermentation product was quantitatively or semi-quantitatively determined using a rapid protein detection method to assess its protein yield potential.

[0024] The miniaturized culture system is a 96-well deep-well plate, a 384-well microplate, or a microfluidic chip culture system; the rapid protein detection method is a dye-binding method, a near-infrared spectroscopy method, or a biosensor-based real-time detection method.

[0025] Preferably, in step (3)b), the visual activity screening using a report system based on the functional properties of the target active ingredient includes the following steps:

[0026] The strains are inoculated into specific selection media containing a reporter system and cultured. The ability of the strains to synthesize the target active ingredient can be intuitively and quickly determined by the visual signals generated by the reporter system.

[0027] The reporting system is based on chemical colorimetric reactions, fluorescent reporter systems, or microbial indicator bacteria.

[0028] When the target active ingredient is a polysaccharide, the reporter system is a phenol-sulfuric acid colorimetric system; when the target active ingredient is a polyphenol or terpene with antioxidant activity, the reporter system is a system based on oxidative stress-sensitive fluorescent reporter bacteria; when the target active ingredient is an antibacterial substance, the reporter system is based on the inhibition zone method of pathogen indicator bacteria.

[0029] The visualization signal is at least one of color change, fluorescence intensity, and inhibition zone size.

[0030] The second objective of this invention is to provide a re-evaluation and screening kit for edible and medicinal fungi, comprising a miniaturized culture device, a rapid protein detection reagent, and a specific reporter system culture medium plate or reagent for visual screening of one or more target active ingredients.

[0031] Preferably, the report system culture medium plate is a plate pre-filled with a phenol-sulfuric acid colorimetric component culture medium, or a plate pre-coated with a reporter bacteria layer.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] 1. High throughput and speed: Utilizing miniaturized culture and rapid detection, along with a visual reporting system, it can process hundreds or thousands of strains simultaneously, greatly increasing screening throughput and shortening the screening cycle from months to weeks using traditional methods.

[0034] 2. Simultaneous Dual-Effect Assessment: For the first time, the assessment of protein yield and specific active ingredient synthesis ability is integrated into the same screening process, which can directly screen strains with high potential for both protein and active ingredient production, avoiding the one-sidedness of traditional single-target screening.

[0035] 3. Clearly targeted: By adding specific precursors to the primary screening medium and using a functional-based reporting system in the secondary screening, the screening process is highly targeted, improving the efficiency and accuracy of screening target strains.

[0036] 4. Balancing safety and cost-effectiveness: A safety initial screening step is introduced to eliminate potentially risky strains in advance. Integrated screening reduces the workload of subsequent invalid validation, saving time and costs. Attached Figure Description

[0037] Figure 1 This is a flowchart of a method for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients, according to the present invention.

[0038] Figure 2 This is a schematic diagram of the visual screening of target active ingredients based on colorimetric reactions in Embodiment 1 of the present invention (a flat panel displays different color depths).

[0039] Figure 3 This is a schematic diagram of the operation of the miniaturized deep-well plate culture and high-throughput protein detection device of the present invention;

[0040] Figure 4 a is a bar chart comparing the protein yield of the superior Ganoderma lucidum strains screened in Example 1 of this invention with that of traditional strains;

[0041] Figure 4 b is a bar chart comparing the yield of Ganoderma lucidum polysaccharides between the superior Ganoderma lucidum strains screened in Example 1 of this invention and traditional strains.

[0042] Figure 5 a is a schematic diagram of the visual screening and inoculation of antibacterial activity based on reporter bacteria in Embodiment 3 of the present invention;

[0043] Figure 5 b is a schematic diagram of the visual screening results of antibacterial activity based on reporter bacteria in Example 3 of the present invention. Detailed Implementation

[0044] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings. It should be understood that the preferred embodiments described herein are for illustration and explanation only and are not intended to limit the present invention.

[0045] Example 1: Screening of Ganoderma strains that produce high levels of protein and Ganoderma polysaccharides.

[0046] 1. Primary screening: 100 Ganoderma lucidum strains were inoculated onto PDA screening plates with soybean meal as the main nitrogen source and supplemented with small amounts of glucose and magnesium sulfate, and incubated at 25°C for 7 days. 50 strains with rapid growth, dense and white mycelium, and neat edges were selected for the next round of screening.

[0047] 2. Initial screening for safety: Observe the colonies of the above 50 strains and remove 2 strains that produced unexplained brown exudate. Use PCR to detect common fungal toxin synthesis-related genes (such as primers related to the tr gene cluster), remove 1 positive strain, leaving 47 strains.

[0048] 3. Secondary review and screening:

[0049] a) Protein potential assessment: Mycelial blocks of 47 strains were inoculated into 24-well plates containing 2 ml of liquid culture medium (main carbon source: corn cob powder; nitrogen source: soybean meal powder) and cultured at 25°C and 180 rpm for 5 days with shaking. Mycelia were harvested, dried, weighed, and crude protein content was rapidly determined using the Bradford method. 25 strains with a protein content higher than 20% (dry weight) were preliminarily screened.

[0050] b) Visual screening of polysaccharide synthesis potential: such as Figure 2 As shown, a solid screening medium containing phenol-sulfuric acid reagent as the main component was prepared. The above 25 bacterial strains were inoculated onto this plate and cultured for 5 days. Then, concentrated sulfuric acid was sprayed evenly onto the plate surface. Due to the color reaction between Ganoderma lucidum polysaccharides and phenol-sulfuric acid reagent, a brownish-red ring appeared around the colony. The darker the color and the larger the ring diameter, the stronger the polysaccharide production or secretion capacity. Based on this, 10 strains with strong color signals were screened.

[0051] To determine the maximum phenol concentration that would not affect the growth of the strain, a tolerance experiment was conducted. The target Ganoderma lucidum strain was inoculated onto solid culture medium containing gradient concentrations of phenol (0%, 0.05%, 0.1%, 0.2%, 0.5%, 1%) and incubated at 25°C for 7 days, with colony diameter measured daily. The results showed that when the phenol concentration was below 0.2%, the colony growth rate was not significantly different from the control group (0%) (P>0.05); however, growth was significantly inhibited when the concentration reached 0.5% (P<0.05). Therefore, a phenol concentration of 0.1%–0.2% was selected as the safe working concentration for subsequent visual screening, ensuring normal strain growth while providing sufficient reactive groups for later color development.

[0052] Secondly, phenol is a major potential growth inhibitor in the culture medium used for screening plates. Based on literature studies of fungi such as *Aspergillus cristatus* and *Phellinus linteus*, phenol is typically used in the reaction as a 5% aqueous solution. To avoid affecting growth, it needs to be diluted.

[0053] Concentrated sulfuric acid has strong dehydrating and oxidizing properties and can instantly kill cells. Therefore, it must never be added directly to the growth medium. First, culture the bacterial strain on a medium containing a low concentration of phenol until the colonies mature. After culturing, spray or pour concentrated sulfuric acid (such as 98% H2SO4) onto the plate surface. This step is crucial for initiating the colorimetric reaction. Since the sulfuric acid is added later, it does not affect the activity of the bacterial strain during its growth phase and therefore does not need to be diluted in the medium.

[0054] 4. Performance Verification: These 10 candidate strains were subjected to small-scale fermentation in a 5L fermenter. After fermentation, mycelial biomass, protein content, and Ganoderma polysaccharide content in the fermentation broth and mycelium were measured. Ultimately, an optimal strain, Ganoderma-XX, was obtained, with a mycelial protein content of 35% (dry weight), representing a 75% increase in protein content compared to traditional production strains, and a 120% increase in Ganoderma polysaccharide yield compared to traditional production strains (see...). Figure 4 ).

[0055] Example 2: Screening for Poria cocos strains that produce high levels of protein and triterpenoids.

[0056] 1. Primary screening: 80 strains of Poria cocos were inoculated onto modified malt extract medium plates supplemented with lanosterol (a precursor for triterpenoid synthesis) and cultured at 28°C for 10 days. 40 strains with good growth were then selected.

[0057] 2. Initial screening for safety: Microscopic examination of mycelial morphology was performed, and 3 strains suspected of contamination or containing abnormal spores were removed, leaving 37 strains.

[0058] 3. Secondary review and screening:

[0059] a) Protein potential assessment: Deep-well plate culture was used similarly to that in Example 1. Near-infrared spectroscopy was used to quickly scan the lyophilized bacterial powder. Based on the established protein calibration model, the protein content was predicted, and 20 strains with high protein potential were screened out.

[0060] b) Screening for Triterpenoid Synthesis Potential: A reporter system based on antioxidant activity was constructed. Strains were seeded on soft agar plates containing a reporter *E. coli* strain sensitive to oxidative stress (carrying the green fluorescent protein gene, whose expression is regulated by oxidative stress inducers). *Poria cocos* triterpenoids possess antioxidant activity, reducing oxidative stress in the surrounding environment and thus inhibiting the fluorescent expression of the reporter bacteria. After cultivation, observation under a fluorescence microscope revealed strains exhibiting a fluorescence-inhibiting zone around the colony, indicating a stronger triterpenoid production capacity. Fifteen strains with obvious fluorescence inhibition zones were screened.

[0061] 4. Performance Verification: Shake-flask fermentation was performed on 15 strains. The contents of major triterpenoids, such as pachymic acid, were determined by HPLC, and the protein content was also determined. A strain of Poria cocos, Porta-YY, was obtained, with a protein content of 28% and a total triterpenoid yield that was 90% higher than the control.

[0062] Example 3: Screening of Cordyceps strains with high protein and antibacterial activity.

[0063] 1. Primary screening: Cordyceps strains are inoculated into a culture medium containing insect peptone, and strains with rapid growth are screened.

[0064] 2. Safety screening: Abnormal sporulation strains are eliminated through morphological screening.

[0065] 3. Secondary review and screening:

[0066] a) Protein potential assessment: Same as in Example 1.

[0067] b) Screening for antibacterial activity: The double-layer plate method was used. For example... Figure 5 As shown, the lower layer is a solid culture medium for Cordyceps militaris, inoculated with candidate strains. The upper layer is covered with soft agar inoculated with reporter pathogens (such as Staphylococcus aureus). After cultivation, if the Cordyceps militaris strains can produce antimicrobial active substances, a clear inhibition zone will appear around their colonies. Strains with strong activity are screened based on the diameter of the inhibition zone.

[0068] 4. Performance verification: Verify the protein yield of the candidate strains and extract the active substances for antibacterial spectrum and minimum inhibitory concentration determination.

[0069] Example 4: A general method for screening edible and medicinal fungi that produce high levels of protein and β-glucan.

[0070] This embodiment aims to establish a universal screening method applicable to various edible and medicinal fungal genera (such as Ganoderma, Lentinus, Schizophyllum, etc.) to simultaneously obtain superior strains with high mycelial protein content and high cell wall / fermentation broth β-glucan yield.

[0071] 1. Primary screening (based on specific carbon sources and substrates)

[0072] This step aims to utilize the differentiated design of culture medium components to initially enrich strains that have an advantage in utilizing specific carbon sources and initially demonstrate the potential for β-glucan synthesis.

[0073] Culture medium preparation: A modified solid screening medium was prepared, with the following basic components: yeast extract 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, and agar 20 g / L. To simulate plant fiber raw materials and screen strains that can efficiently utilize non-grain carbon sources, microcrystalline cellulose at 10 g / L was set as the main carbon source, supplemented with 2 g / L dextran as an inducer.

[0074] Inoculation and culture: One hundred different species of edible and medicinal fungi (including Ganoderma lucidum, Lentinus edodes, Trametes versicolor, etc.) were broken into 5 mm diameter mycelial discs and inoculated into the center of the above-mentioned culture medium plates. Four strains were inoculated into each plate, with three replicates. The plates were incubated at 25℃ in the dark for 7-14 days (adjusted according to the growth rate of the strains).

[0075] Screening criteria: The initial screening mainly examines two criteria.

[0076] Growth rate: Colony diameter was measured daily to calculate the average growth rate (mm / day). Strains with a growth rate lower than 50% of the average rate of all tested strains were discarded. This ensured that the strains advancing to the next round had the basic capacity for biomass accumulation, a prerequisite for high protein production.

[0077] Colony morphology and transparency: Observe the colony morphology. Prioritize strains with dense, white mycelia and vigorous aerial hyphae. Simultaneously, observe whether a clear zone appears around the colony. Since the cellulose in this medium is insoluble granules, a clear zone will appear around the colony if the strain can secrete cellulase to decompose it. Prioritize strains with a clear zone diameter to colony diameter ratio (HC value) greater than 1.2. This preliminarily indicates that the strain has a strong ability to degrade macromolecular substrates and may be more suitable for low-cost fermentation substrates.

[0078] Screening results: Based on the above criteria, 40 strains with rapid growth, dense mycelium, and high HC value were selected from 100 strains and entered the initial safety screening.

[0079] 2. Initial safety screening (based on morphological and conserved toxic gene molecular marker detection)

[0080] Since edible and medicinal fungi will be used as raw materials for food or pharmaceuticals, safety assessment is crucial. This step employs a dual verification approach of "phenotype + genotype".

[0081] A. Morphological microscopic observation:

[0082] Procedure: Select a small amount of hyphae from the primary screening qualified strains, place them on a glass slide, add lactic acid phenol cotton blue staining solution, cover with a coverslip, and observe the morphology of hyphae and spores under an optical microscope.

[0083] Screening criteria: Observe whether the hyphae have typical clamp connections (a characteristic of basidiomycetes), whether the cell walls are smooth, and whether there are any abnormal hyphal swelling, twisting, or breakage. More importantly, check for the presence of suspicious toxin-producing structures. Discard any strains with abnormal hyphal morphology or those showing suspected pathogenic fungal contamination (such as typical conidial heads of Penicillium or Aspergillus). Typically, this step will eliminate about 5-10% of abnormal or contaminated strains.

[0084] B. Molecular toxicity marker detection:

[0085] Genomic DNA extraction: Collect a small amount of fresh hyphae and extract total DNA using a fungal genomic DNA extraction kit.

[0086] PCR detection: PCR amplification was performed on conserved toxin synthesis-related genes in common toxin-producing fungi (especially some closely related species) among edible and medicinal fungi.

[0087] Detection targets: Design degenerate or specific primers to detect conserved domains of the fungal toxin polyketide synthase gene (PKS) and the nonribosomal peptide synthase gene (NRPS). These two gene families are involved in the biosynthesis of most fungal toxins (such as aflatoxin, ochratoxin, etc.).

[0088] PCR system: 25 μL reaction system, containing 50 ng template DNA, 1 μL each of forward and reverse primers (10 μM), 12.5 μL 2×TaqPCR Master Mix, and ddH2O to 25 μL.

[0089] Reaction program: 94℃ pre-denaturation for 5 minutes; 94℃ denaturation for 30 seconds, 55℃ annealing for 30 seconds, 72℃ extension for 1 minute, for a total of 35 cycles; 72℃ final extension for 10 minutes.

[0090] Screening criteria: PCR products were subjected to agarose gel electrophoresis. Any strain that amplified a band of the expected size (indicating the presence of a potential toxin-synthesizing gene fragment), regardless of band strength, was discarded. For strains with unclear bands, sequencing was performed for confirmation.

[0091] Screening results: Combining morphological and molecular marker detection, 5 strains were removed from 40 strains (3 strains were morphologically suspicious, and 2 strains were positive for PKS / NRPS gene detection), and the remaining 35 safe strains entered the re-evaluation screening.

[0092] 3. Secondary screening (simultaneous evaluation of protein and β-glucan)

[0093] a) Protein yield potential assessment: The same deep-well plate miniaturized culture system as in Example 1 was used. Mycelial homogenates of 35 safe strains were inoculated into 24-well deep-well plates containing 2 mL of optimized liquid culture medium (30 g / L corn flour, 20 g / L soybean meal, 10 g / L wheat bran) and cultured at 25°C with shaking at 200 rpm for 6 days. Mycelia were harvested, freeze-dried, and weighed. The Bradford method was used to rapidly determine the soluble protein content, and the total protein yield was calculated based on the biomass. Strains with a protein content higher than 25% (dry weight) or ranking in the top 20 in total protein yield were preliminarily screened.

[0094] b) Visual Screening of β-glucan Synthesis Potential: Visual screening was performed using the fluorescent dye Aniline Blue, which specifically binds to β-(1,3)-D-glucan. Aniline Blue stock solution, filtered and sterilized, was added to sterilized solid culture medium (PDA or optimized medium) to a final concentration of 0.01% (w / v). High-protein-potential strains were inoculated onto this fluorescent screening plate, with three replicates per strain, and incubated at 25°C in the dark for 7 days. After incubation, the plates were observed and photographed using a UV transilluminator or gel imaging system (excitation wavelength 395 nm, emission wavelength 495 nm). Colonies producing high levels of β-glucan emitted strong blue fluorescence around the mycelium or within the colony. The average fluorescence intensity of each colony was quantitatively determined using image analysis software (such as ImageJ). The top 10 strains with the highest fluorescence intensity were selected for further validation.

[0095] 4. Performance Verification and Evaluation

[0096] These 10 candidate strains were scaled up and validated in a 5L fermenter. Biomass, protein content, and amino acid composition were measured. The β-glucan content in the mycelial cell wall and fermentation broth was precisely determined using an enzymatic digestion method combined with HPLC. Finally, the strain with the best overall performance was selected.

[0097] Example 5: Screening for Phellinus linteus strains that produce high levels of protein and laccase (lignin-degrading enzyme).

[0098] This embodiment aims to screen for superior strains of the *Phellinus spp.* that can efficiently produce microbial protein and secrete laccase at high levels. Laccase is a copper-containing polyphenol oxidase that can degrade lignin and has important applications in the feed industry (improving feed digestibility), bioenergy (pretreatment of lignocellulose raw materials), and environmental protection (treatment of phenol-containing wastewater). Screening for *Phellinus spp.* strains with high laccase production can achieve the dual integration of "protein feed production" and "lignocellulose raw material degradation".

[0099] 1. Primary screening (based on the colorimetric reaction of the lignin model compound ABTS).

[0100] This step utilizes the property that laccase can oxidize specific substrates to produce color changes, and preliminarily screens strains with laccase secretion ability from a large number of Phellinus linteus strains.

[0101] Culture medium preparation: A solid screening medium containing the lignin model compound ABTS (2,2'-azido-bis-3-ethylbenzothiazoline-6-sulfonic acid) was prepared. The basal medium formulation was: glucose 10 g / L, peptone 2 g / L, yeast extract 1 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, and agar 20 g / L. After sterilization, when the medium cooled to approximately 50°C, filtered and sterilized ABTS stock solution was added to achieve a final ABTS concentration of 0.03% (w / v). ABTS itself is colorless or light green and, upon oxidation by laccase, generates stable blue-green cationic free radicals.

[0102] Inoculation and culture: Sixty preserved strains of Phellinus linteus from different sources (such as Phellinus baum and Phellinus fissura) were broken into 6 mm diameter mycelial discs and inoculated into the center of the ABTS screening plates. Three strains were inoculated into each 90 mm plate, with three replicates. The plates were incubated at 26°C in the dark for 5-10 days.

[0103] Screening criteria: Observe and record daily whether a blue-green oxidation zone appears around the colonies. Screening is mainly based on the following two criteria:

[0104] Color development time: Record the number of days required from inoculation to the appearance of a visible blue-green ring. Prioritize strains with early color development (e.g., within 3-5 days), as this indicates rapid laccase synthesis and strong secretion capacity.

[0105] Color intensity and ring diameter ratio: At the end of cultivation (e.g., day 7), measure the colony diameter (d) and the diameter of the blue-green oxidation zone (D). Calculate the ring diameter ratio (R = D / d). Simultaneously, visually assess the depth of the blue-green color or use a colorimeter. Prioritize strains with an R value greater than 1.5 and a deep green color.

[0106] Screening results: Based on the above criteria, 25 strains with early color development, large circle diameter ratio, and deep color were selected from 60 strains and entered the initial safety screening.

[0107] 2. Initial safety screening (morphological observation and risk assessment of heavy metal enrichment capacity)

[0108] Since most Phellinus linteus strains originate from the wild or different preservation institutions, and may be used in feed or food-related fields in the future, their safety must be rigorously assessed. This step, in addition to routine morphological observation, specifically includes a risk assessment of their heavy metal accumulation capacity.

[0109] A. Morphological microscopic observation:

[0110] Procedure: Select a small amount of hyphae from the primary screening qualified strains, place them on a glass slide, add lactic acid phenol cotton blue staining solution, and observe the hyphal morphology and reproductive structure under an optical microscope.

[0111] Screening criteria: Observe whether the hyphae have clamp connections (a typical feature of basidiomycetes such as Phellinus linteus), whether the hyphal cell walls are smooth, and whether there are abnormal hyphal swelling, curling, or abnormal granules in the cell contents. At the same time, check for contamination characteristics of other fungi (such as Penicillium and Aspergillus). Discard any strains with severely abnormal hyphal morphology or obvious contamination.

[0112] B. Risk assessment of heavy metal enrichment capacity:

[0113] Principle: Some macrofungi have an extremely strong ability to accumulate heavy metals (such as lead, cadmium, mercury, and arsenic). Even if these strains are not toxic themselves, if the raw materials contain excessive levels of heavy metals, the products may pose a safety hazard. Therefore, strains with lower heavy metal accumulation capabilities should be prioritized.

[0114] Procedure: The 25 strains that passed the initial screening were inoculated into liquid culture media containing gradient concentrations of heavy metal ions for tolerance testing. The following were added to the basal liquid culture medium:

[0115] Lead ions (Pb) 2+ ): 0, 5, 10, 20 mg / L (in the form of Pb(NO3)2)

[0116] Cadmium ions (Cd) 2+ ): 0, 0.5, 1, 2 mg / L (in CdCl2 form)

[0117] The strain was inoculated into culture media containing different concentrations of the aforementioned heavy metals and cultured at 26°C with shaking for 7 days. After culture, the mycelium was collected by filtration, dried, and weighed, and the relative growth rate was calculated (biomass at each concentration / biomass of the blank control × 100%). Simultaneously, a portion of the mycelium was collected, digested, and the enrichment of heavy metals within the mycelium was determined using atomic absorption spectrometry or ICP-MS.

[0118] Screening criteria:

[0119] Growth inhibition rate: Selected at moderate concentrations of heavy metals (such as Pb) 2+ 10 mg / L, Cd 2+ Even at 1 mg / L, the relative growth rate of the strain can still be maintained at over 70%, ensuring that it has a certain degree of production tolerance.

[0120] Enrichment coefficient: Calculate the enrichment coefficient (concentration of heavy metals in the bacterial cell / initial concentration of heavy metals in the culture medium). Prioritize strains with enrichment coefficients below the average level (e.g., below 5). Strains with abnormally high enrichment coefficients (e.g., >10), even if they have high laccase activity, should be discarded or marked as high-risk.

[0121] Screening results: Based on comprehensive morphological and heavy metal risk assessment, 4 strains were removed from the 25 strains (1 strain had abnormal morphology, and 3 strains had excessive heavy metal accumulation capacity), leaving 21 safe strains for re-evaluation and screening.

[0122] 3. Re-evaluation and screening (simultaneous assessment of protein yield potential and laccase activity)

[0123] a) Protein yield potential assessment:

[0124] Miniaturized culture: Mycelial homogenate (or mycelial cake) of 21 safe strains was inoculated into 24-well deep-well plates containing 3 mL of liquid enzyme-producing medium. The medium formulation was as follows: wheat bran 20 g / L (providing inducer and nutrients), soybean meal 15 g / L, glucose 5 g / L, potassium dihydrogen phosphate 1 g / L, magnesium sulfate 0.5 g / L, and copper sulfate 0.1 g / L (copper ions are the cofactor of laccase and can induce laccase synthesis).

[0125] Culture conditions: Cultured at 26℃ and 200 rpm for 8 days with shaking.

[0126] Sample processing: After cultivation, the fermentation broth was transferred to centrifuge tubes and centrifuged at 8000 rpm for 10 minutes at 4°C to separate the supernatant (extracellular enzyme solution) and the cell pellet. The cell pellet was washed twice with distilled water, freeze-dried, and weighed (recording the biomass).

[0127] Rapid protein content detection: Take a small amount of lyophilized bacterial powder and determine the soluble protein content using the Bradford method or BCA method kit. Combine this with biomass to calculate the total protein yield (mg / well). Select the top 15 strains in terms of total protein yield to proceed to the next step of laccase activity screening. If the biomass of a strain is too low (below 50% of the average of all strains), it will be discarded even if the protein content is high.

[0128] b) Visual screening of laccase activity:

[0129] Principle: This method utilizes the specific substrate of laccase to develop color on a plate, allowing direct observation of the enzyme production capacity of the strain. Besides ABTS, substrates such as guaiacol or α-naphthol can also be used.

[0130] Screening plate preparation: Prepare a solid culture medium with the same basic components as the liquid enzyme-producing medium (add 20 g / L agar). After sterilization, cool to approximately 50°C, add a filtered and sterilized 0.04% guaiacol solution (laccase can oxidize guaiacol to produce a reddish-brown compound), mix well, and then pour the plate.

[0131] Inoculation and culture: The above 15 high-protein potential strains were inoculated on this guaiacol screening plate and cultured at 26℃ for 5-7 days.

[0132] Screening criteria:

[0133] Color zone diameter: Observe whether a reddish-brown oxidation zone appears around the colony during cultivation. Measure the diameter (H) of the oxidation zone at the end of cultivation (e.g., day 7). Screen strains with an oxidation zone diameter (H) greater than 15 mm.

[0134] Quantitative confirmation of enzyme activity: For strains with a large oxidation zone, the laccase activity of their liquid fermentation supernatant can be measured simultaneously. Laccase activity assay method: Using 0.5 mM ABTS as substrate, in acetate-sodium acetate buffer at pH 4.5, the rate of change of absorbance is measured at 420 nm (ε = 36000 M). -1 cm -1 One unit of enzyme activity (U) is defined as the amount of enzyme required to oxidize 1 μmol of ABTS per minute.

[0135] Screening results: Based on the determination of oxidation zone diameter and / or enzyme activity, five strains with the highest laccase activity were selected for performance verification.

[0136] 4. Performance Verification and Evaluation

[0137] Scale-up fermentation validation: Five candidate strains were subjected to small-scale fermentation in a 5L fermenter. The same liquid enzyme-producing medium as in the re-evaluation stage was used, and the aeration rate and stirring speed were controlled for 7-10 days. Biomass, mycelial protein content, and laccase activity in the fermentation supernatant were measured periodically.

[0138] Product Function Evaluation:

[0139] Protein nutritional value evaluation: The amino acid composition of the mycelial protein of the optimal strain was determined, the essential amino acid index (EAAI) was calculated, and its potential as a protein feed ingredient was evaluated.

[0140] Verification of laccase's ability to degrade lignin: Fermentation supernatant (crude enzyme solution) was collected and reacted with crushed corn stalks or wheat stalks under suitable conditions. The changes in lignin content in the samples before and after treatment were measured (using the Klason lignin method or acetyl bromide method), as well as the improvement in enzymatic hydrolysis saccharification rate, to verify its application potential in actual lignocellulose degradation.

[0141] Final results: Based on comprehensive evaluation of protein yield, laccase activity, and actual degradation effect, the optimal strain of Phellinus LL was selected. This strain has a mycelial protein content of over 30%, and its fermentation broth laccase activity reaches over 5000 U / L. Furthermore, after treating corn straw with its crude enzyme solution, the lignin degradation rate increased by 25%, and the cellulose enzymatic hydrolysis saccharification rate increased by 40%, demonstrating excellent application prospects of "one strain, two effects".

[0142] Comparative Example 1: Traditional single-target screening method (screening only proteins).

[0143] One hundred Ganoderma strains from Example 1 were used. No initial screening, culture medium optimization, or safety screening was performed. All strains were directly subjected to shake-flask fermentation (three replicates per strain, totaling 300 flasks). After 10 days of cultivation, mycelium was harvested, dried, and the biomass was weighed. The protein content was then precisely determined using the Kjeldahl method. The entire process took approximately six weeks and was extremely labor-intensive. Ultimately, one strain, Ganoderma-A, was selected with the highest protein content. However, subsequent testing revealed that the Ganoderma polysaccharide yield of this strain was only average.

[0144] Comparative Example 2: Traditional single-target screening method (screening only active ingredients).

[0145] Eighty strains of Poria cocos from Example 2 were used. Protein potential assessment was not performed. All strains were directly subjected to shake-flask fermentation. After 14 days of culture, mycelium was collected, extracted with organic solvents, and the triterpenoid content was analyzed by HPLC. The entire process took approximately eight weeks and was costly. Ultimately, a strain named Porta-B with the highest triterpenoid yield was selected. However, subsequent analysis revealed that its mycelial biomass and protein content were low, resulting in poor overall economic efficiency.

[0146] Comparative Example 3: The method of the present invention omits the screening of the visualization report system.

[0147] The procedure of Example 1 was adopted, but in the re-evaluation screening step b), phenol-sulfuric acid visualization screening was not used. Instead, all 25 high-protein potential strains were subjected to shake-flask fermentation, and their polysaccharide content was determined one by one using the phenol-sulfuric acid method. Although this method could ultimately find strains with high yields in both ways, it increased the workload of 25 shake-flask fermentations and chemical detections, prolonging the re-evaluation phase by 2 weeks and resulting in the loss of throughput advantage.

[0148] Performance Comparison: Compared with Comparative Examples 1 and 2, the method of this invention can simultaneously obtain information on high yields of both protein and active ingredients within a similar or shorter timeframe, resulting in strains with superior overall performance. Compared with Comparative Example 3, this invention, by introducing a visualization reporting system, significantly improves the throughput and speed of assessing the synthetic potential of active ingredients during the re-evaluation stage, demonstrating the integrated innovation advantages of this invention.

[0149] like Figure 3As shown, the protein yield potential assessment in step (3)a) of this invention is based on an integrated high-throughput micro-fermentation and rapid detection platform. This platform consists of three parts working collaboratively: an automated liquid handling workstation 31, a 96-well plate 32, and a rapid protein detection device 33. The automated workstation 31 inoculates the test strain into the 96-well plate 32 for parallel fermentation; after fermentation, the 96-well plate 32 is transferred to the rapid protein detection device 33; the data collected by the rapid protein detection device 33 is used to guide subsequent screening. Its typical operation flow and data transfer relationship are as follows:

[0150] First, automated inoculation and dispensing (workstation → deep well plate).

[0151] First, the seed culture or mycelial homogenate of several edible and medicinal fungal strains that passed the initial safety screening in step (2) are placed in the source plate positions of an automated liquid handling workstation (such as Tecan Freedom EVO or Hamilton Microlab STAR). Using a pre-programmed script, the workstation's multi-channel pipette tip can simultaneously and precisely aspirate equal volumes of bacterial culture from the source plate and inoculate them into each well of a 96-well deep-well plate (each well typically containing 1-2 mL of working volume). Simultaneously, the workstation can automatically dispense sterile fermentation medium into each well. This process enables parallel and rapid inoculation of hundreds of samples, avoiding the low throughput and contamination risks associated with manual operation.

[0152] Second, miniaturized parallel fermentation (deep-well plate → shaking incubator).

[0153] After inoculation and dispensing, the 96-well deep-well plates were sealed with a breathable sealing film and transferred to a multi-layer shaking incubator for isothermal shaking culture. The deep-well plate design allows for high-throughput micro-fermentation within a limited space. Its special lid and vent design ensure gas exchange, enabling the strains in each well to grow in a relatively uniform microenvironment, thereby obtaining comparable biomass accumulation and protein expression data. This is the core element of the "miniaturized culture" of this invention.

[0154] Third, high-throughput data acquisition (deep well plate → rapid protein detection equipment).

[0155] After fermentation, the 96-well plate is transferred to a rapid protein detection device used in conjunction with a workstation or operated independently. This device can be:

[0156] Multifunctional microplate reader: When used with Bradford and other colorimetric reagents, it directly reads the absorbance of each well at 595 nm and quickly calculates the relative protein content. Near-infrared (NIR) spectrometer: Equipped with a microplate scanning accessory, it can directly perform non-contact scanning of lyophilized bacterial powder or fermentation broth, and output protein content data of all samples in all wells within seconds based on a pre-established quantitative model.

[0157] Fourth, data integration and screening decisions (testing equipment → data analysis system).

[0158] The high-throughput data generated by the rapid protein detection device (such as the strain number and protein content corresponding to each well) is automatically transmitted to the data analysis system (usually the software or bioinformatics analysis platform that comes with the workstation). The system automatically generates a candidate list of strains with high protein potential based on preset thresholds (such as the top 30% of protein content) to guide the visual screening of active ingredients in step (3)b).

[0159] In summary, Figure 3 This invention illustrates how it leverages the precise sample loading capability of an automated workstation, the high-throughput culture capability of deep-well plates, and the parallel reading capability of rapid detection equipment. By organically combining these three elements through the physical flow of samples (deep-well plates) and the information flow of data (from workstation commands to detection results), it forms a complete high-throughput protein yield potential assessment system. This integrated application of the system is a key feature that distinguishes this invention from traditional low-throughput shake-flask screening.

[0160] Finally, it should be noted that the above descriptions are merely preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients, characterized in that, Includes the following steps: Step (1): Primary screening: The edible and medicinal fungal strains to be tested are inoculated on the primary screening medium and cultured. Preliminary screening is carried out based on the colony growth rate and morphological uniformity. Step (2): Safety screening: For the strains obtained from the initial screening, safety assessment is performed based on colony morphology characteristics and / or molecular toxicity markers to eliminate potentially unsafe strains; Step (3): Re-evaluation and screening: For strains that pass the initial safety screening, the following two types of evaluations are performed in parallel or sequentially: a) Assess protein yield potential using miniaturized culture and rapid protein detection methods; b) Visual activity screening using a reporting system based on the functional properties of the target active ingredient; Step (4): Performance verification and evaluation: Fermentation verification and product evaluation are carried out on the candidate strains obtained from the re-evaluation.

2. The method for screening edible and medicinal fungi that produce high levels of microbial protein and target active ingredients according to claim 1, characterized in that, In step (1), the edible and medicinal fungal strain is at least one of the following: Ganoderma, Poria, Cordyceps, Phellinus, Hericium, and Lentinus. The primary screening culture medium contains specific precursor substances that are beneficial to the biosynthesis of the target active ingredient.

3. The method for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients according to claim 2, characterized in that, The target active ingredient is at least one of polysaccharides, terpenoids, polyphenols, sterols, and antibacterial proteins. The specific precursor substances include at least one of lanosterol, phenylalanine, coumaric acid, mevalonic acid, sucrose, and amino acids.

4. The method for screening edible and medicinal fungi that produce high levels of microbial protein and target active ingredients according to claim 1, characterized in that, In step (1), the primary screening medium contains one or more non-synthetic nitrogen sources.

5. The method for screening edible and medicinal fungi that produce high levels of microbial protein and target active ingredients according to claim 4, characterized in that, The non-synthetic nitrogen sources include soybean meal, cottonseed meal, corn steep liquor powder, yeast extract, or insect peptone.

6. The method for screening edible and medicinal fungi that produce high levels of microbial proteins and target active ingredients according to claim 1, characterized in that, In step (2), the safety assessment of colony morphology characteristics includes whether abnormal pigments are produced and whether the hyphal morphology is normal; the safety assessment using molecular toxicity markers includes molecular marker detection based on toxin-related genes.

7. The method for screening edible and medicinal fungi that produce high levels of microbial protein and target active ingredients according to claim 1, characterized in that, In step (3)a), assessing protein yield potential using miniaturized culture and rapid protein detection methods includes the following steps: The strain was inoculated into a miniaturized culture system for liquid fermentation, and the protein content of the fermentation product was quantitatively or semi-quantitatively determined using a rapid protein detection method to assess its protein yield potential. The miniaturized culture system is a 96-well deep-well plate, a 384-well microplate, or a microfluidic chip culture system; the rapid protein detection method is a dye-binding method, a near-infrared spectroscopy method, or a biosensor-based real-time detection method.

8. The method for screening edible and medicinal fungi that produce high levels of microbial protein and target active ingredients according to claim 1, characterized in that, In step (3)b), the visual activity screening using a report system based on the functional properties of the target active ingredient includes the following steps: The strains are inoculated into specific selection media containing a reporter system and cultured. The ability of the strains to synthesize the target active ingredient can be intuitively and quickly determined by the visual signals generated by the reporter system. The reporting system is based on chemical colorimetric reactions, fluorescent reporter systems, or microbial indicator bacteria. When the target active ingredient is a polysaccharide, the reporter system is a phenol-sulfuric acid colorimetric system; when the target active ingredient is a polyphenol or terpene with antioxidant activity, the reporter system is a system based on oxidative stress-sensitive fluorescent reporter bacteria; when the target active ingredient is an antibacterial substance, the reporter system is based on the inhibition zone method of pathogen indicator bacteria. The visualization signal is at least one of color change, fluorescence intensity, and inhibition zone size.

9. A re-evaluation and screening kit for edible and medicinal fungi for implementing the method as described in any one of claims 1-8, characterized in that, This includes miniaturized culture devices, rapid protein detection reagents, and specific reporter system culture media plates or reagents for visual screening of one or more target active ingredients.

10. The edible and medicinal fungi re-evaluation screening kit according to claim 9, characterized in that, The reporting system culture medium plates are plates pre-filled with phenol-sulfuric acid chromogenic medium or plates pre-coated with reporter bacteria.

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