Preparation method of composite microbial agent and application of composite microbial agent in resisting heavy-cropping stress

Abstract

The application belongs to the technical field of soil improvement, and discloses a preparation method of a composite microbial agent and application of the composite microbial agent in resisting heavy-cropping stress. Active components of the composite microbial agent are Clonostachys rosea, Trichoderma harzianum and Pseudomonas chlororahs, and a carrier contains a basic carrier, a synergistic component and soil adaptive components (oyster shell powder or earthworm manure particles). The preparation comprises the following steps: strain fermentation, carrier construction, solid-state co-fermentation, drying and mixing of the fermented product and the carrier. After the composite microbial agent is used, the abundance of Clonostachys rosea, Trichoderma harzianum and Pseudomonas chlororahs in the soil of heavy-cropping crops obviously increases, and the abundance of Fusarium oxysporum significantly decreases; the acidification of acidified soil is obviously improved, the content of organic acids (ferulic acid and vanillic acid) in the soil obviously decreases, the soil becomes looser from being hardened, and the soil is beneficial to the improvement of root activity and the resistance to diseases and pests; the composite microbial agent can also significantly improve single fruit weight and sugar content, and reduce the storage rot rate of fruits, and the effect is remarkable.

Description

Technical Field

[0001] This invention belongs to the field of soil improvement technology and relates to a method for preparing a compound microbial agent and its application in resisting continuous cropping stress. Background Technology

[0002] Replant stress, also known as replanting obstacle or replanting disease, is prevalent in horticulture, cash crop, and food crop production. With economic development, the demand for various fruits, vegetables, and other agricultural products has increased dramatically, leading to a rapid expansion of crop planting areas and making replanting inevitable. Replant stress has become one of the biggest challenges restricting agricultural development.

[0003] Apple cultivation has a long history, and Shaanxi Province, with its favorable climate and soil and hydrological conditions, has become one of the main apple-producing areas. Currently, as apple trees age, most orchards are entering their senescent stage, leading to serious replanting stress problems. Therefore, a deeper understanding of the mechanisms underlying apple replanting stress and finding methods or products to alleviate and overcome apple replanting diseases is of great significance to the healthy and sustainable development of the apple industry.

[0004] Currently, the main control measures for apple replanting stress include soil disinfection, rootstock selection, and biological control. Soil disinfection primarily includes physical disinfection and chemical fumigation; however, the overuse of chemical fumigants can severely pollute the local environment, damage the soil microecology, and make subsequent remediation extremely difficult. Rootstock selection mainly involves using appropriate combinations of scions and rootstocks from different species to increase fruit tree yield and enhance the tree's resistance to various biotic and abiotic stresses. Biological control mainly utilizes the competitive or antagonistic effects of beneficial microorganisms in the soil against pathogens to reduce the incidence of replanting diseases, thereby mitigating the adverse effects of replanting stress.

[0005] Studies have shown that continuous cropping stress significantly alters the rhizosphere soil microbial community, with a sharp decrease in the relative abundance of bacteria and a dramatic increase in the relative abundance of fungi. Therefore, from the perspective of regulating the distribution of plant rhizosphere soil microorganisms, exogenous application of microbial agents will help transform the soil microbial community from a fungal to a bacterial type, increase the relative abundance of beneficial bacteria, inhibit the growth and reproduction of pathogens, and thus effectively alleviate continuous cropping stress in apples. However, currently, there is a lack of mature and effective microbial agents for alleviating continuous cropping stress in apples. Existing measures for alleviating continuous cropping stress are often accompanied by environmental pollution or excessive costs. Therefore, the development of compound microbial agents that can alleviate continuous cropping obstacles in apples is urgently needed. Summary of the Invention

[0006] To improve soil health and reduce apple yield decline caused by continuous cropping obstacles, this invention provides an application of a compound microbial agent in resisting continuous cropping stress. The active ingredients of the compound microbial agent are: Trichoderma harzianum, Trichoderma harzianum, and Pseudomonas aeruginosa.

[0007] The compound microbial agent also includes a carrier, the components of which are: 90% basic carrier, 9.0% synergistic component, and 1.0% soil-adaptive component by mass percentage.

[0008] 90% of the base carrier contains: 40% bentonite, 30% humic acid, and 20% biochar;

[0009] The 9.0% synergistic component contains: 8.5% porous starch microspheres, 0.3% chitosan oligosaccharide, 0.1% trehalose, and 0.1% γ-aminobutyric acid;

[0010] The soil-adaptive components are: oyster shell powder or earthworm castings granules.

[0011] Furthermore, in the above applications, the oyster shell powder is activated by calcination at 800℃, with a fineness of <200 mesh, making it suitable for acidified soils; earthworm castings granules are suitable for compacted soils.

[0012] Furthermore, in the above applications, the crop includes apples.

[0013] On the other hand, the present invention provides a method for preparing a compound microbial agent, comprising: strain fermentation, carrier construction, solid-state co-fermentation, and preparation of the compound microbial agent;

[0014] The active ingredients of the compound microbial agent are: Trichoderma harzianum, Trichoderma harzianum, and Pseudomonas aeruginosa;

[0015] The compound microbial agent also includes a carrier, the components of which are: 90% basic carrier, 9.0% synergistic component, and 1.0% soil-adaptive component by mass percentage.

[0016] 90% of the base carrier contains: 40% bentonite, 30% humic acid, and 20% biochar;

[0017] The 9.0% synergistic component contains: 8.5% porous starch microspheres, 0.3% chitosan oligosaccharide, 0.1% trehalose, and 0.1% γ-aminobutyric acid;

[0018] The soil-adaptive components are: oyster shell powder or earthworm castings granules.

[0019] Furthermore, in the above preparation method, the fermentation of the microorganisms includes: *Trichoderma harzianum* fermentation, *Pseudomonas aeruginosa* fermentation, and *Pseudomonas aeruginosa* fermentation, wherein:

[0020] The fermentation medium for *Aureobasidium* was: 30 g / L corn flour, 15 g / L soybean meal, and 0.1% chitosan oligosaccharide, with the remainder being water, sterilized at high temperature; the fermentation conditions were: 25℃ for 72 h, with dissolved oxygen >40%, until the spore concentration was ≥5×10⁻⁶. 10CFU / mL;

[0021] The fermentation medium for Trichoderma harzianum is as follows: 200g of potato chunks are boiled in water and the liquid is retained. 20g of wheat bran and 2g of chitin microcrystals are added, and water is added to make up to 1L. The mixture is then sterilized at high temperature. The fermentation conditions are: fermentation at 28℃ for 48h with intermittent stirring until the chitinase activity is ≥200U / mL.

[0022] The fermentation medium for *Pseudomonas aeruginosa* was: 20 g / L sucrose, 10 g / L peptone, and 0.01% FeSO4, sterilized at high temperature; the fermentation conditions were: pH=7.0±0.2, fermented at 30℃ for 24 h, until the phenazine yield was ≥100 mg / L.

[0023] Furthermore, in the above preparation method, the carrier construction includes: mixing 40% bentonite, 30% humic acid, 20% biochar, 8.5% porous starch microspheres, 0.3% chitosan oligosaccharide, 0.1% trehalose, 0.1% γ-aminobutyric acid, and 1% oyster shell powder or earthworm castings granules evenly by mass percentage.

[0024] Furthermore, in the above preparation method, the solid-state co-fermentation includes:

[0025] 1) Preparation of symbiotic fermentation medium: glucose 20g / L, peptone 5g / L, ammonium nitrate 3g / L, K2HPO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4 0.01g / L, chitosan oligosaccharide 0.3g / L, yeast extract 2g / L, pH=6.5±0.2;

[0026] 2) Symbiotic fermentation: After sterilization, the symbiotic fermentation medium was inoculated with Pseudomonas aeruginosa at an inoculation rate of 1 wt%, and fermented with shaking at 26-30°C for 24 hours. Then, 2 wt% of Trichoderma harzianum was inoculated, and fermentation was continued with shaking for another 24 hours. After inoculation with Trichoderma harzianum, 1 wt% of Trichoderma harzianum was inoculated, and fermentation was continued for another 24 hours after inoculation with Trichoderma harzianum.

[0027] Furthermore, in the above preparation method, the preparation of the composite microbial agent includes: drying the fermented material after solid-state co-fermentation at low temperature, and then mixing it with a carrier, wherein the mixing ratio is: carrier: dried fermented material = 100: 5~10 by mass.

[0028] Furthermore, in the above preparation method, the total number of effective viable bacteria in the compound microbial agent is ≥5×10⁻⁶. 10 CFU / g, number of miscellaneous bacteria <1×10 5 CFU / g.

[0029] Compared with the prior art, the present invention, "a method for preparing a composite microbial agent and its application in resisting continuous cropping stress," has the following beneficial effects:

[0030] In terms of formulation selection, this invention uses *Trichoderma harzianum*, *Pseudomonas aeruginosa*, and *Pseudomonas aeruginosa* as compound active ingredients, combined with a carrier adaptation strategy of oyster shell powder and earthworm castings, thereby improving the soil microbial community structure, soil physicochemical properties, and fruit quality in continuously cropped apple orchards. Oyster shell powder continuously releases calcium carbonate, helping to regulate the pH value of acidic soils, while earthworm castings improve soil permeability and reduce soil compaction. Bentonite, humic acid, and biochar provide adsorption carriers and living space for microorganisms, and also help retain water and fertilizer. Chitosan oligosaccharides help block the production of trichomycin by *Trichoderma harzianum*, trehalose can be used to neutralize the mycotoxins of *Trichoderma harzianum*, and γ-aminobutyric acid has a significant promoting effect on these microorganisms.

[0031] Regarding the improvement in efficacy, the results of using the compound microbial agent provided by this invention show that:

[0032] 1) Soil physicochemical properties: The acidification of acidified soil is significantly improved, the content of organic acids (ferulic acid, vanillic acid) in the soil is reduced, and the soil becomes looser from compaction, which is conducive to improving root vitality and resisting diseases and pests.

[0033] 2) Microbial Community: Beneficial functional bacteria in the soil proliferated, with increased abundance of *Trichoderma harzianum*, *Pseudomonas aeruginosa*, and *Pseudomonas aeruginosa*, while the abundance of *Fusarium oxysporum* decreased. *Trichoderma harzianum*, through mechanisms such as nutrient competition, hyperparasitism, antagonism, and induction of plant resistance, inhibited the activity of various pathogens, effectively reducing the incidence of fruit tree diseases and pests. Chitinase secreted by *Trichoderma harzianum* can decompose the cell walls of pathogenic fungi, thereby inhibiting their growth and reproduction and protecting fruit trees from disease. Phenidine compounds produced by *Pseudomonas aeruginosa*, such as phenazine-1-carboxylic acid and 2-hydroxyphenazine, have significant inhibitory effects on plant pathogenic fungi. These compounds can destroy the cell membranes of pathogenic fungi, preventing their growth and reproduction, thus protecting plants from disease.

[0034] 3) Fruit quality: Increased single fruit weight and sugar content, and reduced storage rot rate.

[0035] The compound microbial agent effectively improves the soil microbial community. These microorganisms decompose organic matter to generate humic colloids, which significantly improves the soil aggregate structure, enhances soil aeration and water and fertilizer retention capacity, and alleviates compaction problems. At the same time, the organic acids and enzymes secreted by these microorganisms can decompose insoluble phosphorus, potassium and other elements in the soil, converting ineffective nutrients into effective forms that can be absorbed by the roots, improving fertilizer utilization and helping to improve fruit quality. Detailed Implementation

[0036] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0037] Unless otherwise specified, the test methods in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0038] The sources of the bacterial strains in the examples are as follows:

[0039] Trichoderma harzianum: purchased from Jinan Binhai Trading Co., Ltd., the finished mycelial powder was cultured to obtain the experimental mycelial cells;

[0040] Broom mold: The described broom mold is pink broom mold, with accession number CGMCC NO.17070, and was obtained as a gift;

[0041] *Pseudomonas aeruginosa*: Preservation number CGMCC NO.26997, sourced as a gift.

[0042] Example 1

[0043] This embodiment describes the preparation of a compound microbial agent.

[0044] 1. Microbial fermentation

[0045] Fermentation method of *Azolla oblongata*: Culture medium components: 30 g / L corn flour, 15 g / L soybean meal, and 0.1% chitosan oligosaccharide, with the remainder being water. The culture medium was sterilized at 121℃ for 30 min, then inoculated with 1% *Azolla oblongata*, and fermented at 25℃ for 72 h, with dissolved oxygen >40%, until the spore concentration was ≥5 × 10⁻⁶. 10 CFU / mL; after fermentation, the spore concentration was measured to be 7 × 10⁻⁶. 10 CFU / mL.

[0046] Fermentation method of *Trichoderma harzianum*: Culture medium composition: Boil 200g of potato chunks in water, retain the liquid, add 20g of wheat bran and 2g of chitin microcrystals, and add water to a final volume of 1L. Sterilize the culture medium at 121℃ for 30min, inoculate with 2% *Trichoderma harzianum*, and ferment at 28℃ for 48h with intermittent stirring until the chitinase activity is ≥200U / mL; after fermentation, the chitinase activity was measured to be 268U / mL.

[0047] Fermentation method of Pseudomonas aeruginosa: 20 g / L sucrose, 10 g / L peptone and 0.01% FeSO4, pH=7.0. The culture medium was sterilized at 121℃ for 30 min, and 1% Pseudomonas aeruginosa was inoculated. Fermentation was carried out at 30℃ for 24 h until the phenazine yield was ≥100 mg / L. After fermentation, the phenazine content was measured to be 141 mg / L.

[0048] 2. Carrier Construction

[0049] Oyster shell powder calcination: Oyster shell powder is activated by calcination at 800℃ for 4 hours, and then ground to a fineness of <200 mesh after cooling.

[0050] By weight percentage, 1600g (40%) of bentonite, 1200g (30%) of humic acid, 800g (20%) of biochar, 340g (8.5%) of porous starch microspheres, 12g (0.3%) of chitosan oligosaccharide, 4g (0.1%) of trehalose, 4g (0.1%) of γ-aminobutyric acid, and 40g (1.0%) of oyster shell powder were mixed evenly to obtain 4000g of carrier.

[0051] 3. Solid-state co-fermentation

[0052] 1) Prepare the symbiotic fermentation medium: glucose 20g / L, peptone 5g / L, ammonium nitrate 3g / L, K2HPO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4 0.01g / L, chitosan oligosaccharide 0.3g / L, yeast extract 2g / L, pH=6.5.

[0053] 2) Symbiotic fermentation: After sterilizing the symbiotic fermentation medium at 121℃ for 30 min, Pseudomonas aeruginosa was inoculated at a bacterial inoculation rate of 1 wt%, and fermented at 28℃ with shaking for 24 h. Then, Trichoderma harzianum was inoculated, and fermentation continued with shaking for another 24 h. Finally, Trichoderma harzianum was inoculated, and fermentation continued for another 24 h after inoculation with Trichoderma harzianum.

[0054] 4. Preparation of compound microbial agents

[0055] After solid-state co-fermentation, the fermented material was dried at 4°C and pulverized to obtain a powdered solid, which was then mixed with a carrier. The mixing ratio was 4000g of carrier and 200g of dried fermented material to obtain a compound microbial agent. The total effective viable count of the compound microbial agent was 8 × 10⁻⁶. 10 CFU / g, bacterial count 1×10⁻⁶ 4 CFU / g.

[0056] Example 2

[0057] This embodiment describes the preparation of a compound microbial agent.

[0058] The preparation method of the compound microbial agent is the same as in Example 1, except that the mixing ratio of the fermentation product and the carrier is: 4000g of carrier and 400g of dried fermentation product to obtain the compound microbial agent.

[0059] Example 3

[0060] This embodiment describes the preparation of a compound microbial agent.

[0061] The preparation method of the compound microbial agent is the same as in Example 1, except that earthworm castings of the same mass are used instead of oyster shell powder to obtain the compound microbial agent.

[0062] Example 4

[0063] This embodiment describes the preparation of a compound microbial agent.

[0064] The preparation method of the compound microbial agent is the same as in Example 1, except that: earthworm castings granules of the same mass are used instead of oyster shell powder; the mixing ratio of fermentation material and carrier is: 4000g carrier and 400g dried fermentation material to obtain the compound microbial agent.

[0065] Example 5

[0066] This embodiment describes a field trial of the compound microbial agent improving the soil microbial community of continuously cropped crops.

[0067] Experimental groups and treatment methods: Acidified soil group 1 (pH=4.5~5.5): treated with the compound microbial agent prepared in Example 1; Acidified soil group 2 (pH=4.5~5.5): treated with the compound microbial agent prepared in Example 2; Compacted soil group 1 (bulk density >1.5g / cm³) 3 ): The compound microbial inoculant prepared in Example 3 was used; 2 groups of compacted soil (bulk density > 1.5 g / cm³) 3 The experimental group used the compound microbial inoculant prepared in Example 4; the control group did not use any inoculant. Five plants were replicated in each group, and both the experimental and control groups received the same water and fertilizer management.

[0068] Experimental site: Yangling, Shaanxi Province; apple variety: Fuji; soil bulk density: 1.55 g / cm³ 3 Porosity 30%, pH=5.1, organic matter 0.9%.

[0069] Experimental method: Select apple planting plots that have been planted in the same location repeatedly. In mid-April, mix the compound microbial agent with the top 0-30cm soil at a rate of 400g / plant and apply it as a base fertilizer. In June, during the fruit expansion period, apply it as a top dressing at a rate of 200g / acre by trenching, while simultaneously moistening it with drip irrigation.

[0070] Indicator testing: 1) Soil physicochemical indicators: pH, bulk density, organic acids (ferulic acid, vanillic acid); 2) Microbial community: plate count method to detect microorganisms in soil samples; 3) Fruit quality: single fruit weight, sugar content, storage decay rate (the percentage of fruits that rotted after 3 months of storage). All the above indicators were tested after fruit harvest.

[0071] Experimental results:

[0072] 1) Soil improvement effect: The results of soil physicochemical index testing are shown in Table 1.

[0073] Table 1. Soil physicochemical properties of the experimental and control groups

[0074]

[0075] 2) Microbial community: The results of the detection of changes in microbial community abundance are shown in Table 2.

[0076] Table 2. Microbial content in the experimental and control groups

[0077]

[0078] 3) Fruit quality: The results of fruit quality testing are shown in Table 3.

[0079] Table 3. Fruit quality of the experimental and control groups

[0080]

[0081] In summary, this invention prepares a compound microbial agent based on *Trichoderma harzianum*, *Pseudomonas aeruginosa*, and *Pseudomonas aeruginosa*, and combines it with a carrier adaptation strategy of oyster shell powder (suitable for acidified soils) and earthworm castings (suitable for compacted soils), significantly improving the soil microbial community structure in continuously cropped apple orchards. Using the compound microbial agent provided by this invention, the results show that: 1) Soil physicochemical properties: The acidification of acidified soils is significantly improved, the content of organic acids (ferulic acid, vanillic acid) in the soil decreases, and the soil becomes looser, which is beneficial for improving root vitality and resisting pests and diseases. 2) Microbial community: Beneficial functional bacteria in the soil proliferate, the abundance of *Trichoderma harzianum*, *Pseudomonas aeruginosa*, and *Pseudomonas aeruginosa* increases, while the abundance of *Fusarium oxysporum* decreases. 3) Fruit quality: The single fruit weight and sugar content of the experimental group increased, and the storage decay rate decreased. The above results indicate that the compound microbial agent provided by this invention effectively improves the soil microbial community. These microorganisms decompose organic matter to generate humic colloids, significantly improving soil aggregate structure, enhancing soil aeration and water and fertilizer retention capacity, and alleviating compaction problems. At the same time, the organic acids and enzymes secreted by these microorganisms can decompose insoluble phosphorus, potassium and other elements in the soil, converting ineffective nutrients into effective forms that can be absorbed by the roots, improving fertilizer utilization and helping to improve fruit quality.

[0082] The embodiments described above are only some, not all, of the embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art through related deductions and substitutions based on the inventive concept, without inventive effort, are within the scope of protection of the present invention.

Claims

1. The application of a compound microbial inoculant in resisting continuous cropping stress, characterized in that, The active ingredients of the compound microbial agent are: Trichoderma harzianum, Trichoderma harzianum, and Pseudomonas aeruginosa; The *Pseudomonas pulmonarius* is *Pseudomonas pulmonarius*, with accession number CGMCC NO.17070; the *Pseudomonas aeruginosa* has accession number CGMCC NO.26997. The compound microbial agent also includes a carrier, the components of which are: 90% basic carrier, 9.0% synergistic component, and 1.0% soil-adaptive component by mass percentage. 90% of the base carrier contains: 40% bentonite, 30% humic acid, and 20% biochar; The 9.0% synergistic component contains: 8.5% porous starch microspheres, 0.3% chitosan oligosaccharide, 0.1% trehalose, and 0.1% γ-aminobutyric acid; The soil-suitable components are: oyster shell powder or earthworm castings granules; the oyster shell powder is activated by calcination at 800℃, has a fineness of <200 mesh, and is suitable for acidified soils; Earthworm castings are suitable for compacted soil; The compound microbial agent is used to combat continuous cropping stress in apples; The preparation method of the compound microbial agent includes: strain fermentation, carrier construction, solid-state co-fermentation, and preparation of the compound microbial agent, wherein the solid-state co-fermentation includes: 1) Preparation of symbiotic fermentation medium: glucose 20g / L, peptone 5g / L, ammonium nitrate 3g / L, K2HPO4 1.5g / L, KH2PO4 0.5g / L, MgSO4·7H2O 0.5g / L, FeSO4 0.01g / L, chitosan oligosaccharide 0.3g / L, yeast extract 2g / L, pH=6.5±0.2; 2) Symbiotic fermentation: After sterilization, the symbiotic fermentation medium was inoculated with Pseudomonas aeruginosa at an inoculation rate of 1 wt%, and fermented with shaking at 26-30°C for 24 hours. Then, 2 wt% of Trichoderma harzianum was inoculated, and fermentation was continued with shaking for another 24 hours. After inoculation with Trichoderma harzianum, 1 wt% of Trichoderma harzianum was inoculated, and fermentation was continued for another 24 hours after inoculation with Trichoderma harzianum.

2. The application according to claim 1, characterized in that, The fermentation process includes: *Trichoderma harzianum* fermentation, *Pseudomonas aeruginosa* fermentation, and *Pseudomonas aeruginosa* fermentation, wherein: The fermentation medium for *Pseudomonas aeruginosa* was: 30 g / L corn flour, 15 g / L soybean meal, and 0.1% chitosan oligosaccharide, with the remainder being water. The medium was sterilized at 121°C for 30 min. The fermentation conditions were: 25°C for 72 h, with dissolved oxygen >40% during this period. The fermentation medium for Trichoderma harzianum is as follows: 200g of potato chunks are boiled in water and the liquid is retained. 20g of wheat bran and 2g of chitin microcrystals are added, and water is added to make up to 1L. The mixture is then sterilized at 121℃ for 30min. The fermentation conditions are: fermentation at 28℃ for 48h with intermittent stirring. The fermentation medium for *Pseudomonas aeruginosa* was: 20 g / L sucrose, 10 g / L peptone, and 0.01% FeSO4, sterilized at 121℃ for 30 min; the fermentation conditions were: pH = 7.0 ± 0.2, fermented at 30℃ for 24 h.

3. The application according to claim 1, characterized in that, The carrier construction includes: mixing 40% bentonite, 30% humic acid, 20% biochar, 8.5% porous starch microspheres, 0.3% chitosan oligosaccharide, 0.1% trehalose, 0.1% γ-aminobutyric acid, and 1% oyster shell powder or earthworm castings granules evenly by weight percentage.

4. The application according to claim 1, characterized in that, The preparation of the compound microbial agent includes: drying the fermented material after solid-state co-fermentation at a low temperature of 4°C, and then mixing it with a carrier. The mixing ratio is: carrier: dried fermented material = 100: 5~10 by mass.

5. The application according to claim 1, characterized in that, The total effective viable count of the compound microbial agent is ≥5×10⁻⁶. 10 CFU / g, number of miscellaneous bacteria <1×10 5 CFU / g.

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