Complex microbial inoculant for dairy product washing water treatment, method and application of complex microbial inoculant

Abstract

The invention belongs to the technical field of resources and environmental microorganisms, and discloses a complex microbial inoculant for dairy product washing water treatment, a method and application of the complex microbial inoculant, and the complex microbial inoculant comprises kluyveromyces marxianus ART-27 and lactobacillus fermentum. The lactobacillus fermentum and the kluyveromyces marxianus adopted in the invention are edible safe strains, and have higher biocompatibility and application safety. Under the culture condition of low-concentration lactose and protein, the compound bacterium system shows a remarkable synergistic effect, and the OD600 value reaches 2.43, which is respectively improved by 76.17% and 51.02% compared with that of single-strain lactic acid bacteria and saccharomycetes fermentation in a control group. 81% of lactose and 94.26% of protein can be utilized when the compound bacteria are used for fermenting the simulated water. The COD (Chemical Oxygen Demand), the TN (Total Nitrogen) and the SS (Suspended Solid) of the actual flushing water can be effectively reduced by fermenting the actual flushing water through the compound bacteria, and the removal rates respectively reach 78.23 percent, 80.67 percent and 84.09 percent.

Description

Technical Field

[0001] This invention belongs to the field of resource and environmental microbiology technology, and in particular to a compound microbial agent, method and application for the treatment of dairy product rinsing water. Background Technology

[0002] The dairy industry generates a large amount of process water during production, and globally, approximately 4 to 11 million tons of dairy waste are discharged into the environment annually, posing a serious threat to biodiversity. Statistics show that processing one liter of milk produces about 6 to 10 liters of wastewater. This wastewater mainly consists of low concentrations of protein, lactose, inorganic salts, nitrogen, phosphorus, and other nutrients, characterized by large discharge volumes and high organic content. Given the current water scarcity and increasingly stringent environmental protection requirements, how to effectively treat and recycle dairy wastewater has become an urgent problem for the industry.

[0003] Currently, the physical treatment of dairy washing wastewater generally adopts a "mixed treatment of wastewater from different processes" model. This "mixed treatment - discharge to meet standards" model mixes high-quality rinsing water rich in lactose and protein with other wastewater, resulting in the dilution and waste of nutrient resources. This not only loses its recycling value but also increases the energy consumption and cost of end-of-pipe treatment. If the treatment of this wastewater could be transformed from a simple "degradation and removal" to "resource-based value-added processing," it would provide the dairy industry with a new path that combines environmental protection and economic benefits. Various bacteria (such as Bacillus cereus, Bacillus subtilis, and Escherichia coli) can be naturally isolated from dairy wastewater, and microorganisms such as Staphylococcus aureus, Fusarium, and Aspergillus can also be obtained through cultivation. These microbial communities have a certain effect on reducing the organic load of wastewater. However, existing microbial agents are generally not listed in the edible fungi species list, and their activity is poor in low-concentration wastewater. The fermentation metabolites are uncertain, limiting the utilization pathways of the treated recycled water. Due to the low concentration of organic matter in dairy product rinsing water, the utilization and metabolic capacity of single microorganisms in dairy water components are limited. Dual-strain co-fermentation, through metabolic complementarity, synergistic effects, and niche differentiation, holds promise for overcoming the bottleneck of slow growth and low utilization rates of single microorganisms under low substrate concentrations. Therefore, constructing complex strains that efficiently utilize low-concentration dairy components is a crucial issue in the resource utilization of dairy water, possessing significant practical and academic research value.

[0004] Therefore, the efficient degradation of dairy wastewater by edible microorganisms to broaden the utilization of recycled water is a technical problem that urgently needs to be solved. It has important research value and application prospects in the field of industrial wastewater treatment and resource utilization. Summary of the Invention

[0005] The purpose of this invention is to overcome the shortcomings of the prior art and provide a compound microbial agent, method and application for dairy water treatment.

[0006] A compound microbial agent for treating dairy product rinsing water, said compound microbial agent comprising Kluyveromyces macrocephala ( Kluyveromyces marxianus ART-27 and Lactobacillus fermentum ( Lactobacillus fermentum ); The taxonomic name for *Kluyveromyces martensii* ART-27 is: *Kluyveromyces martensii* ( Kluyveromyces marxianus The accession number is CGMCC No.39119, the accession date is January 7, 2026, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0007] Furthermore, the fermenting lactobacillus includes Lactobacillus fermentum AF 7-2, Lactobacillus fermentum AF1-3, Lactobacillus fermentum AF1-5, Lactobacillus fermentum ATCC 9338, Lactobacillus fermentum CECT5716, etc. Furthermore, the volume ratio of the Lactobacillus fermentum to Kluyveromyces ART-27 is 1:0.5-1:3, preferably 1:1.

[0008] The application of the compound microbial agent described above in the treatment of low-concentration dairy water.

[0009] Furthermore, the compound bacteria can utilize low concentrations of dairy products to rinse away lactose and protein in the water through fermentation; Alternatively, the dairy water may include dairy wastewater, dairy pipeline flushing water, dairy tank flushing water, and whey.

[0010] The preparation method of the compound microbial agent as described above includes the following steps: (1) Prepare Lactobacillus fermentation seed culture and Kluyveromyces ART-27 seed culture respectively; (2) Wash the two seed cultures from step (1) with 10×PBS, repeat three times, and then adjust to OD. 600 A bacterial suspension with a value of 1; (3) The two bacterial suspensions obtained in step (2) are mixed in a volume ratio of 1:0.5-1:3 to prepare a compound bacterial suspension, thus obtaining a compound bacterial agent.

[0011] Further, in step (1), the method for preparing the fermented Lactobacillus seed liquid is as follows: the strain is activated by streaking, a single colony is picked and inoculated into MRS liquid medium, and then incubated at 37°C for 12-14 h. Further, in step (1), the method for preparing the Kluyveromyces ART-27 seed liquid is as follows: the strain of Kluyveromyces ART-27 is activated by streaking, a single colony is picked and inoculated into YPD liquid medium, and then incubated at 30°C for 16-18 h.

[0012] The method for treating low-concentration dairy product rinsing water using the compound microbial agent described above includes the following steps: The compound microbial agent is inoculated into the low-concentration rinsing water of the dairy products to be treated for fermentation.

[0013] Furthermore, the total inoculation amount of the compound microbial agent is 0.5-7% of the volume of the low-concentration dairy product rinsing water.

[0014] The advantages and positive effects of this invention are as follows: 1. Compared with existing fermentation rinsing water treatment microbial agents, the *Lactobacillus fermentum* and *Kluyveromyces martensii* strains used in this invention are both edible and safe strains, exhibiting higher biocompatibility and application safety. Under low-concentration lactose and protein culture conditions, this composite microbial system demonstrates a significant synergistic effect, with OD... 600 The value reached 2.43, which was 76.17% and 51.02% higher than the single strain of lactic acid bacteria and yeast fermentation in the control group, respectively.

[0015] 2. The β-galactosidase activity of the compound bacteria of the present invention can reach a maximum of 2.95 U / ml at 48 h, which is 24.41% higher than that of single strain Kluyveromyces martensii and 72.88% higher than that of Lactobacillus fermentum.

[0016] 3. The present invention significantly reduces the content of residual dairy components in the simulated water fermentation broth, and the compound bacteria utilize 81% of the lactose and 94.26% of the protein in approximately 48 hours of fermentation. Furthermore, when the compound bacteria are added to actual rinsing water, the compound bacteria (ART-27 / AF 7-2) utilize 70%-81.76% of the reducing sugar and 80%-88.44% of the protein.

[0017] 4. The present invention uses simulated water as a fermentation base. Its components can be treated with compound bacteria to reduce the content of bacterial protein in the rinsing water of dairy products to 0.63 g / L, with a protein content ratio of 53.08%. The bacterial protein content is increased by 45.6% and 64.32% respectively compared with single-strain fermentation.

[0018] 5. Both Lactobacillus AF 7-2 and Kluyveromyces Marcius ART-27 of this invention are listed in the "List of Microbial Strains that Can Be Used in Food" published by the National Health Commission of China. They do not produce toxic or harmful substances and can be used as safe strains for food in the fermentation of dairy product rinsing water, which facilitates the recycling of water.

[0019] 6. The compound microbial agent of this invention exhibits a significant synergistic effect in treating low-concentration dairy product rinse water, efficiently degrading lactose and protein. Simultaneously, this compound microbial agent effectively reduces COD, TN, and SS in the actual rinse water, achieving removal rates of 78.23%, 80.67%, and 84.09%, respectively. This invention provides a safe, efficient, and economical new approach for the resource-based treatment of dairy product wastewater. Attached Figure Description

[0020] Figure 1 The fermenting lactobacillus AF 7-2 in this invention ( Lactobacillus fermentum ) and Max Kluyveromyces ( Kluyveromyces marxianus Colony morphology images and electron microscope images of ART-27; among them, Figure 1 A and C are colony morphology diagrams and electron microscope images of AF 7-2; Figure 1 B and D are colony morphology diagrams and electron microscope images of ART-27; Figure 2 This is a comparison chart of the specific growth rate (right figure) and cell biomass (left figure) of the compound bacteria and single bacteria in this invention; Figure 3 This is a comparison diagram of the β-galactosidase activity of the compound bacteria and single bacteria in this invention; Figure 4 This is a control diagram illustrating the fermentation and utilization of lactose and protein in simulated water in this invention. Figure 5 This is a comparison image of the batter rinse water after fermentation and utilization (left image) and the untreated water (right image) in this invention.

[0021] The classification name of Kluyveromyces martinis ART-27 described in this invention is: Kluyveromyces martinis ( Kluyveromyces marxianus The accession number is CGMCC No.39119, the accession date is January 7, 2026, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), No.3, No.1 Beichen West Road, Chaoyang District, Beijing. Detailed Implementation

[0022] The present invention will be further described below with reference to the embodiments. The following embodiments are descriptive and not limiting, and should not be used to limit the scope of protection of the present invention.

[0023] The various experimental operations involved in the specific embodiments are all conventional techniques in the field. For parts not specifically annotated in this document, those skilled in the art can refer to various commonly used reference books, scientific and technological documents or related instructions and manuals prior to the filing date of this invention to carry out the operations.

[0024] A compound microbial agent for treating dairy product rinsing water, said compound microbial agent comprising Kluyveromyces macrocephala ( Kluyveromyces marxianus ART-27 and Lactobacillus fermentum ( Lactobacillus fermentum ); The taxonomic name for *Kluyveromyces martensii* ART-27 is: *Kluyveromyces martensii* ( Kluyveromyces marxianus The accession number is CGMCC No.39119, the accession date is January 7, 2026, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0025] Furthermore, the Lactobacillus fermentum includes Lactobacillus fermentum AF 7-2, Lactobacillus fermentum AF1-3, Lactobacillus fermentum AF1-5, Lactobacillus fermentum ATCC 9338, and Lactobacillus fermentum CECT5716; Alternatively, the volume ratio of the Lactobacillus fermentum and Kluyveromyces ART-27 is 1:0.5-1:3.

[0026] Furthermore, the volume ratio of the Lactobacillus fermentum and Kluyveromyces ART-27 is 1:1.

[0027] The application of the compound microbial agent described above in the treatment of low-concentration dairy water.

[0028] Furthermore, the compound bacteria can utilize low concentrations of dairy products to rinse away lactose and protein in the water through fermentation; Alternatively, the dairy water may include dairy wastewater, dairy pipeline flushing water, dairy tank flushing water, and whey.

[0029] (1) Prepare Lactobacillus fermentation seed culture and Kluyveromyces ART-27 seed culture respectively; (2) Wash the two seed cultures from step (1) with 10×PBS, repeat three times, and then adjust to OD. 600 A bacterial suspension with a value of 1; (3) The two bacterial suspensions obtained in step (2) are mixed in a volume ratio of 1:0.5-1:3 to prepare a compound bacterial suspension, thus obtaining a compound bacterial agent.

[0030] Further, in step (1), the method for preparing the fermented Lactobacillus seed liquid is as follows: the strain is activated by streaking, a single colony is picked and inoculated into MRS liquid medium, and then incubated at 37°C for 12-14 h. Further, in step (1), the method for preparing the Kluyveromyces ART-27 seed liquid is as follows: the strain of Kluyveromyces ART-27 is activated by streaking, a single colony is picked and inoculated into YPD liquid medium, and then incubated at 30°C for 16-18 h.

[0031] The method for treating low-concentration dairy product rinsing water using the compound microbial agent described above includes the following steps: The compound microbial agent is inoculated into the low-concentration rinsing water of the dairy products to be treated for fermentation.

[0032] Furthermore, the total inoculation amount of the compound microbial agent is 1-5% of the volume of the low-concentration dairy product rinsing water.

[0033] Furthermore, the small molecule substances in dairy product rinsing water include, but are not limited to: lactose (2-10 g / L), protein (2-10 g / L), sulfates (manganese sulfate, magnesium sulfate), and phosphates (dipotassium hydrogen phosphate). In this invention, a simulated water composition was designed with 5 g / L lactose, 5 g / L protein, 0.02 g / L manganese sulfate, 0.2 g / L magnesium sulfate, and 2 g / L dipotassium hydrogen phosphate, using water as the solvent.

[0034] Furthermore, Lactobacillus fermentum AF 7-2 ( Lactobacillus fermentum ) and Max Kluyveromyces ( Kluyveromyces.marxianus ART-27 compound bacteria fermentation utilizes simulated dairy components in water.

[0035] (1) Preparation of seed culture: The lactic acid bacteria were taken out of the preservation strain from the 4℃ refrigerator and activated by streak plate twice. Single colonies were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37℃ for 12 h. After centrifugation at 4℃ and 6000 r / min for 5 min, the supernatant was discarded. The culture was washed 3 times with 10×PBS to remove residual nutrients in the liquid medium and resuspended in an equal volume of 10×PBS.

[0036] Yeast cultures were removed from the 4°C freezer and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL YPD liquid medium in test tubes and incubated at 30°C for 16 h. The cultures were then centrifuged at 4°C and 6000 r / min for 5 min, the supernatant was discarded, and the cultures were washed three times with 10×PBS to remove residual nutrients from the YPD liquid medium. The cultures were then resuspended in an equal volume of 10×PBS.

[0037] (2) Dilute the viable counts of lactic acid bacteria and yeast to OD600≈1. Add a total inoculum of 2% (1% lactic acid bacteria and 1% yeast) to a 50 ml shake flask containing fermentation medium. The fermentation medium consists of lactose 5 g / L, protein 5 g / L, manganese sulfate 0.02 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 2 g / L, water as solvent, and adjust the pH to 6.

[0038] (3) Take samples, centrifuge to obtain supernatant, and determine the biomass, viable number, and consumption rate of lactose and protein in the single-strain control group and the double-strain experimental group.

[0039] (4) Weigh the precipitate obtained by centrifugation in step (3), dry it under vacuum freeze drying, weigh it after constant drying, and determine the protein content of the constant-dry bacterial protein by Kjeldahl nitrogen analyzer.

[0040] Furthermore, Lactobacillus fermentum AF 7-2 ( Lactobacillus fermentum ) and Max Kluyveromyces ( Kluyveromyces.marxianus The ART-27 compound bacteria were added to the actual production rinsing water for fermentation. After fermentation, the concentration of residual sugar, residual protein, pH, and viable bacteria in the water were measured.

[0041] Furthermore, the water discharge indicators, after flushing water treatment, were measured for water quality parameters COD (chemical oxygen demand), TN (total nitrogen content), and SS (suspended solids).

[0042] Specifically, the relevant preparation and testing methods are as follows: Example 1: Screening and Combination of Lactic Acid Bacteria 1. Screening of lactic acid bacteria (1) Lactic acid bacteria were inoculated into MRS liquid medium and cultured at 37℃ for 12-14 h. Then, the bacterial culture was spread onto MRSA medium and cultured at 37℃ for 12-14 h. Then, 0.2% X-Gal solution was added dropwise to the lactic acid bacteria colonies and cultured at 37℃ for 48 h, during which the color change of the lactic acid bacteria colonies was observed. When the lactic acid bacteria colonies changed from white to blue, it indicated that the colonies had the ability to produce β-galactosidase. A single blue colony could be picked and inoculated into MRS liquid medium and cultured at 37℃ for 12-14 h. Finally, 30% glycerol was added and stored at -80℃. The selected strains had a strong ability to produce β-galactosidase and a strong ability to decompose and utilize lactose. According to the X-Gal rapid screening method, a total of 97 lactic acid bacteria strains in the laboratory strain bank were screened, and 33 strains of lactic acid bacteria were selected.

[0043] (2) The lactic acid bacteria obtained in step (1) were added to 10 g / L protein and cultured for 24 h to obtain 9 strains with biomass OD. 600 ≥1 strain.

[0044] (3) The lactic acid bacteria obtained in step (2) were inoculated into a culture medium with low concentrations of lactose (5 g / L) and protein (5 g / L) as the sole carbon and nitrogen source (5 g / L lactose, 5 g / L protein, 0.02 g / L manganese sulfate, 0.5 g / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, and water as the solvent). The medium was then statically cultured at 37°C for 12-14 h to obtain 3 strains with biomass OD. 600 ≥1 strain (3 strains of Lactobacillus fermentum) Lactobacillus fermentum (AF 1-3, AF 1-5, AF 7-2), these three strains are all well-known strains in the prior art, and have been published in the article Niu Jiwei. Isolation, Identification and Diversity Analysis of Lactic Acid Bacteria in the Brewing Process of Shanxi Aged Vinegar [D]. Tianjin University of Science and Technology, 2016.). Three strains of Lactobacillus fermentum (AF 1-3, AF 1-5, AF 7-2) were obtained through screening. Lactobacillus fermentum AF 1-3, AF 1-5, and AF 7-2 were combined with the mutagenized yeast ART-27 to obtain the optimal fermentation combination of Lactobacillus fermentum AF 7-2 and ART-27.

[0045] 2. Kluyveromycin ( Kluyveromyces marxianus Acquisition of ART-27 Mutagenesis and selection of high-efficiency strains: ARTP mutagenesis: (1) Activation of the starting strain: The starting strain, Kluyveromyces martensii KM-6, was inoculated into a YPD medium in an Erlenmeyer flask and cultured at 30°C for 24-30 h as the starting strain for ARTP mutagenesis. (2) Preparation of starting bacterial suspension: Take 1 mL of seed liquid from step (1), centrifuge at 12000 r / min for 2 min, discard the supernatant and collect the bacterial cells, wash the bacterial cells 2-3 times with 0.9% physiological saline, resuspend and dilute 100 times to prepare a bacterial suspension with uniform cell dispersion, which will be used as the starting bacterial suspension for subsequent ARTP treatment; (3) ARTP mutagenesis treatment: Take 5-10 μL of the starting bacterial suspension prepared in step (2) and spread it evenly on a sterilized metal slide. Place it in an ARTP mutagenesis breeding instrument for mutagenesis treatment. The ARTP treatment parameters are set as follows: working gas is high-purity helium, radio frequency power is 70-120 W, gas flow rate is 8-12 slm, treatment time is 30-100 s, and irradiation distance is 2-4 mm. (4) Post-mutation culture: Wash off the mutagenized cells with 990 μL of 0.9% sterile physiological saline, dilute appropriately, and spread on a low-concentration carbon and nitrogen source (lactose, protein) solid culture medium plate (lactose 5 g / L, protein 5 g / L, manganese sulfate 0.02 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 2 g / L, agar 2 g / L, solvent is water), and incubate upside down at 28-34℃ for 2-3 days.

[0046] Preservation of highly effective mutagenic bacteria: (1) Observe the colonies obtained in step 1 on solid culture medium, pick a single colony from the plate and inoculate it into YPD medium and incubate at 30°C for 16 h, and store it in a 60% glycerol tube at -80°C. (2) The mutant strain and the original strain obtained in step (1) were streaked in a “z” shape onto YPD slant medium and cultured at 30℃ for 2-3 days.

[0047] A strain of *Kluyveromyces martensii* capable of efficiently utilizing low concentrations of lactose and protein for growth was obtained. The name of this *Kluyveromyces martensii* strain is ART-27, and its taxonomic name is *Kluyveromyces martensii*. Kluyveromyces marxianus The accession number is CGMCC No.39119, the accession date is January 7, 2026, and the depositary institution is the China General Microbiological Culture Collection Center, No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

[0048] The physicochemical characteristics of the lactic acid bacteria AF 7-2 are as follows: Gram-positive bacteria, spherical in shape on solid culture medium, milky white in color (e.g., ...). Figure 1 As shown), the optimal pH is 5-6.5, and the optimal growth temperature is 30℃-37℃. The physicochemical characteristics of yeast ART-27 are as follows: Solid culture medium is spherical and milky white in color (as shown). Figure 1 As shown in the figure, the optimal pH is 4.5-5.5. Using biomass as a growth indicator, the optimal fermentation conditions for dual-strain fermentation were determined. The final co-culture fermentation conditions were determined to be an inoculation ratio of 1:1, a fermentation temperature of 30-34 ℃, an inoculation sequence of simultaneous addition of lactic acid bacteria and yeast, an optimal pH of 6, and a fermentation time of 48 h.

[0049] The Max Kluyveromyces ( Kluyveromyces marxianus The physicochemical properties of ART-27 are as follows: Gram-positive bacteria; colonies on solid culture media are spherical, moist, and milky white in color. Figure 1 As shown in the figure, the optimal growth pH is 4-5 and the optimal growth temperature is 28-34℃.

[0050] 3. Combination of lactic acid bacteria and yeast Three strains of lactic acid bacteria were combined with the mutated *Max Kluyveromyces*. Kluyveromyces marxianus The following steps were taken to combine ART-27 with lactic acid bacteria and yeast for co-culture: Three strains of lactic acid bacteria and yeast were seed cultured with ART-27 for 12-16 hours to obtain the seed culture. Specifically: Lactic acid bacteria were taken from a 4℃ freezer and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37℃ for 12 hours. The culture was then centrifuged at 4℃, 6000 r / min for 5 minutes, the supernatant was discarded, and the culture was washed three times with 10×PBS to remove residual nutrients. The culture was then resuspended in an equal volume of 10×PBS. Yeast was taken from a 4℃ freezer and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL of YPD liquid medium and cultured at 30℃ for 16 hours. The culture was then centrifuged at 4℃, 6000 r / min for 5 minutes, the supernatant was discarded, and the culture was washed three times with 10×PBS to remove residual nutrients. The culture was then resuspended in an equal volume of 10×PBS. The OD was then... 600 The seed culture, with its pH adjusted to 1, was inoculated at a total inoculum ratio of 2% (v / v) (1% each of the two bacterial strains) into 5 mL of fresh culture medium (5 g / L lactose, 5 g / L protein, 0.02 g / L manganese sulfate, 0.5 g / L magnesium sulfate, and 2 g / L dipotassium hydrogen phosphate). The medium was then incubated at 32°C for 24 h. Simultaneously, separate cultures of lactic acid bacteria and yeast were set up as controls. The OD value of the fermentation broth was measured. 600 The value was used to assess cell growth. In a 2% inoculum, 5 mL culture system, yeast and lactic acid bacteria were each inoculated at 1%, while the control group consisted of lactic acid bacteria and yeast alone inoculated at 2%. The combination of AF 7-2 and ART-27 showed the highest biomass and OD. 600 The OD200 of AF1-3 and AF1-5 combined with ART-27 after approximately 48 hours of fermentation reached 1.2, representing increases of 52.04% and 39.73% respectively compared to the control groups fermented alone. The OD200 of AF1-3 and AF1-5 combined with ART-27 after 48 hours of fermentation was also improved. 600 The values ​​were 0.92 and 1.01, respectively. AF 7-2 and ART-27 showed improvements of 23.33% and 15.83% compared to these two combinations. The optimal fermentation combination was Lactobacillus fermentum AF 7-2 and Kluyveromyces martensii ART-27. It can also be seen that Kluyveromyces martensii ART-27 and Lactobacillus fermentum AF 7-2 in the compound of this invention have a synergistic effect, synergistically increasing their biomass, etc.

[0051] The preparation of seed culture when lactic acid bacteria and yeast are added separately is as follows: Lactic acid bacteria were taken from the preservation strain at 4℃ and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL of MRS liquid medium and incubated at 37℃ for 12 h. After centrifugation at 4℃ and 6000 r / min for 5 min, the supernatant was discarded, and the culture was washed three times with 10×PBS to remove residual nutrients. The culture was then resuspended in an equal volume of 10×PBS to allow the OD to adjust. 600 Set the value to 1.

[0052] Yeast cultures were removed from storage at 4°C and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL YPD liquid medium in test tubes and incubated at 30°C for 16 h. The cultures were then centrifuged at 4°C and 6000 r / min for 5 min, the supernatant was discarded, and the cultures were washed three times with 10×PBS to remove residual nutrients from the YPD liquid medium. The cultures were then resuspended in an equal volume of 10×PBS to allow the OD500 to adjust. 600 Set the value to 1.

[0053] Example 2: Growth of the compound bacteria under low concentration of dairy components (1) Activation of strain: Lactic acid bacteria AF7-2 ( Lactobacillus fermentum ) and yeast ( Kluyveromyces.marxianus ART-27 seeds were cultured for 12-16 hours to obtain the seed culture. Specifically, the lactic acid bacteria were taken from the preservation strain at 4℃ and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL of MRS liquid medium and cultured at 37℃ for 12 h. After centrifugation at 4℃ and 6000 r / min for 5 min, the supernatant was discarded, and the culture was washed three times with 10×PBS to remove residual nutrients in the liquid medium. The culture was then resuspended in an equal volume of 10×PBS.

[0054] Yeast cultures were removed from the 4°C freezer and activated twice by streak plating. Single colonies were picked and inoculated into 5 mL YPD liquid medium in test tubes and incubated at 30°C for 16 h. The cultures were then centrifuged at 4°C and 6000 r / min for 5 min, the supernatant was discarded, and the cultures were washed three times with 10×PBS to remove residual nutrients from the YPD liquid medium. The cultures were then resuspended in an equal volume of 10×PBS.

[0055] (2) The activated bacterial strains were washed with 0.9% physiological saline. The washed bacterial solution was inoculated at a rate of 2% (1% lactic acid bacteria and 1% yeast). The control group single bacteria were inoculated at a rate of 2% into 50 ml of simulated rinsing water (5 g / L lactose, 5 g / L protein, 0.02 g / L manganese sulfate, 0.5 g / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, and water as solvent). Samples were taken as 0 h samples, and samples were taken at 12, 24, 36, and 48 h to measure OD. 600 .

[0056] (3) Among them, X tOD at time t 600 X0: OD at time t0 600 t: Sampling time t0: Sampling time.

[0057] (4) Calculate the specific growth rate according to the above formula.

[0058] (5) Results are as follows Figure 2 As shown, the OD of the combined fermentation of lactic acid bacteria AF7-2 and yeast ART-27 was measured after 48 h. 600 The value reached 2.43, which was higher than that of the control group of single-strain lactic acid bacteria (OD). 600 (0.58), yeast (OD) 600 The specific growth rates of the bacteria (1.19%) increased by 76.17% and 51.02%, respectively. When the specific growth rate becomes negative, the bacteria enter the decline phase. The compound bacteria enter the decline phase after 48 hours, lactic acid bacteria AF 7-2 after 24 hours, and yeast ART-27 after 36 hours, extending the water treatment time by at least 12 hours. Therefore, the time for decomposing and utilizing components such as lactose, protein, and inorganic salts in the rinsing water is extended, increasing their component consumption rate. It can also be seen that the Kluyveromyces martensii ART-27 and Lactobacillus fermentum AF 7-2 in the compound of this invention have a synergistic effect, synergistically increasing their specific growth rates, etc.

[0059] Example 3: Utilization of dairy product components by the compound bacteria To evaluate the activity of the compound microbial agent of this invention on key enzymes in lactose metabolism, the β-galactosidase activity was determined using the o-nitrobenzene-β-D-galactosidase (ONPG) method. The specific steps are as follows: Cell pretreatment: The cells were cultured statically at 30-34°C for 60 h in simulated aqueous medium (5 g / L lactose, 5 g / L protein, 0.02 g / L manganese sulfate, 0.5 g / L magnesium sulfate, 2 g / L dipotassium hydrogen phosphate, with water as the solvent). Samples were taken every 12 h from both the experimental group (a co-culture of *Lactobacillus fermentum* AF7-2 and *Kluyveromyces martensii* ART-27 (prepared as in step 1 of Example 2), with AF7-2 and ART-27 each accounting for 1% of the inoculum) and the control group (a single-strain culture, prepared as in step 1 of Example 2). The samples were centrifuged at 8000 × g for 10 minutes at 4°C, and the supernatant was discarded. The resulting cell pellet was washed twice with pre-cooled phosphate-buffered saline (10×PBS, pH 7.0) to remove residual culture medium components. The bacterial pellet was then resuspended in an equal volume of 10×PBS to obtain a crude bacterial suspension.

[0060] (1) Standardization of bacterial suspension: Adjust the concentration of the above crude bacterial suspension to achieve an absorbance (OD) of 560 nm. 560 The concentration was approximately 1.0 ± 0.05, which was used as a standardized bacterial suspension for subsequent enzyme activity assays.

[0061] (2) Cell permeability treatment: Take 1.0 mL of standardized bacterial suspension, add 50 μL of toluene, and shake vigorously on a vortex mixer for 7 minutes to disrupt cell membrane permeability and allow intracellular enzymes to fully contact the substrate.

[0062] (3) Establishment and termination of enzyme reaction system: Take 100 μL of the permeabilized bacterial suspension, add 900 μL of PBS to dilute it, and record the absorbance value at 560 nm at this time, which is recorded as A1. 560 Immediately afterwards, 200 μL of o-nitrobenzene-β-D-galactopyranoside (ONPG) solution (4 g / L, dissolved in PBS) was added, and the mixture was thoroughly mixed. The mixture was then placed in a 37°C water bath and reacted for 15 minutes. After the reaction was complete, 0.5 mL of 1 mol / L Na₂CO₃ solution was quickly added to terminate the reaction.

[0063] (4) Absorbance measurement and enzyme activity calculation: The absorbance of the mixture after the reaction was terminated was measured at 420 nm (A). 420 ) and 560 nm (A2) 560 The absorbance value at ( ). β-galactosidase activity (E a (Unit: U / mL) Calculated using the following formula: in: A 420 : The absorbance of the reaction product o-nitrophenol at 420 nm; A1 560 Initial absorbance of diluted bacterial suspension at 560 nm (reflecting bacterial cell concentration); A2 560 : The absorbance of the mixture after reaction at 560 nm (used to correct for turbidity interference); Reaction time: 15 min in this experiment; The volume (mL) of bacterial suspension added to the reaction system was 1 in this experiment; Coefficient 1.75: This is an empirical correction factor used to eliminate the interference of background turbidity on the experiment (see Miller 1972).

[0064] (5) Calculation and Analysis of Results: The β-galactosidase activity of the compound bacteria and the control group was measured every 12 h. For the compound bacteria, the peak value appeared at 48 h (approximately 2.95 U / ml). For the control group, the peak value of lactic acid bacteria enzyme activity appeared at 24 h (approximately 1.2 U / ml); for yeast KW-6, the peak value appeared at 36 h (approximately 2.45 U / ml). The lactic acid bacteria and yeast in the compound bacteria may have complementary nutrition and metabolic specialization, which promotes the overall enzyme synthesis efficiency. The ability and efficiency of the compound bacteria in utilizing and decomposing the substrate lactose are improved. The results are as follows: Figure 3 As shown.

[0065] 2. Simulate the residual lactose concentration in water To quantitatively evaluate the lactose utilization capacity of the compound microbial agent of this invention, the residual lactose concentration during fermentation was determined using the anthrone colorimetric method. The specific procedure is as follows: (1) Standard curve preparation: Prepare a standard lactose solution with a concentration of 0.1 mg / mL. Take 0.2 mL, 0.4 mL, 0.6 mL, 0.8 mL and 1.0 mL of the standard solution into 5 clean test tubes, and add distilled water to make up to a total volume of 1.0 mL, so that the final lactose concentrations in each tube are 0.02, 0.04, 0.06, 0.08 and 0.10 mg / mL, respectively. Set up a blank control tube and add 1.0 mL of distilled water. Accurately add 4.0 mL of 0.2% (w / v, mass concentration) anthrone-concentrated sulfuric acid reagent (freshly prepared) to each test tube, mix thoroughly, and heat in a boiling water bath for 10 minutes. After removal, quickly cool to room temperature, and measure the absorbance (OD) of each tube at a wavelength of 620 nm. 620 Plotting lactose concentration (mg / mL) on the ordinate (Y) and corresponding OD... 620 The x-axis value is used as the x-axis. Linear regression analysis yields the standard curve equation: Y = 0.8306X + 0.1297(R²). 2 =0.9993).

[0066] (2) Sample processing and determination: The seed culture of the compound bacteria of the present invention was obtained according to the method step (1) described in Example 2 for simulating the residual lactose concentration in water. It was inoculated into a culture medium containing lactose and simulated dairy product rinsing water (lactose 5 g / L, protein 5 g / L, manganese sulfate 0.02 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 2 g / L, solvent: water). It was allowed to ferment statically for 48 h at 30–34℃ and pH 5–7. From the start of fermentation, samples were taken every 12 hours, centrifuged (8000 × g, 10 min, 4℃) to remove the bacterial cells, and the supernatant was appropriately diluted. The anthrone colorimetric reaction was performed according to the above standard curve plotting method, and the OD was measured. 620 value.

[0067] (3) Calculation and analysis of results: Based on the measured sample OD 620 Substitute the values ​​into the standard curve equation to calculate the actual concentration of lactose in the fermentation broth. The results are as follows: Figure 4 As shown, with an initial lactose concentration of 5 g / L, after 48 hours of fermentation, the residual lactose concentration in the compound microbial agent treatment group decreased to 0.95 g / L, and the lactose utilization rate of the compound microorganisms reached 81%. In the control group, the lactose utilization rates after single-strain fermentation by lactic acid bacteria and yeast were 50% and 71.8%, respectively, while the compound microbial agent improved these rates by 31% and 9.2% compared to single-strain fermentation. Therefore, most of the lactose was utilized, laying the foundation for the recycling of rinsing water.

[0068] 3. Simulate the residual protein concentration in water (1) Standard curve plotting: Take 10 mg of bovine serum albumin (BSA) into a 100 mL volumetric flask, add distilled water to dissolve and dilute to the mark to obtain a BSA standard solution with a concentration of 0.1 mg / L. For the standard curve series of concentration solutions, prepare seven clean volumetric flasks. Take 0.5, 1, 2, 4, 6, 8, and 10 mL of the above standard stock solution into each flask, and dilute to volume with water to obtain standards with concentrations of 0.005, 0.01, 0.02, 0.04, 0.06, 0.08, and 0.1 mg / L. Take 1 mL of each standard into a clean test tube, add 4 mL of the above CBB G-250 dye reagent (weigh 50 mg of CBB G-250 in a 1 L volumetric flask, dissolve in 40 mL of 95% ethanol, then add 120 mL of 85% phosphoric acid, and dilute to volume with distilled water), vortex to mix, and react at room temperature for 5 min. Use blank deionized water as a reference solution, and measure the absorbance of the standard group at 595 nm wavelength using a spectrophotometer. Perform regression analysis on the data to obtain the linear regression equation Y = 4.38x + 0.1312, R0. 2 =0.9992 (where Y is the protein concentration and X is the OD). 595 value).

[0069] (2) Sample processing and determination: The seed culture of the compound bacteria of the present invention was obtained according to the method step (1) described in Example 2, and inoculated into a lactose-containing simulated dairy washing water culture medium (lactose 5 g / L, protein 5 g / L, manganese sulfate 0.02 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 2 g / L, solvent: water). It was allowed to ferment statically for 48 h at 30–34℃ and pH 5–7. From the start of fermentation, samples were taken every 12 hours, centrifuged (8000 × g, 10 min, 4℃) to remove the bacterial cells, and the supernatant was appropriately diluted and the protein content of the sample was determined according to the standard curve plotting method described above.

[0070] (3) Calculation and analysis of results: The results are as follows Figure 5 As shown, the remaining protein in the fermentation broth was 0.29 g / L, and the fermentation utilization rate of the compound bacteria was 94.26%. The protein utilization rates of single-strain lactic acid bacteria and yeast fermentation for 48 hours were 82.32% and 85.32%, respectively. The combined bacterial agent increased the protein utilization rate by 11.94% and 8.94% compared to single-strain fermentation. Therefore, compared to lactose, lactic acid bacteria and yeast have a greater advantage in processing and consuming whey protein. Most of the protein was utilized, laying the foundation for the recycling of the rinsing water.

[0071] Example 4: Utilization of Basauce Water Fermented with Compound Bacteria We obtain pasteurized rinse water from dairy companies. This process inactivates the vast majority of pathogenic and putrefactive microorganisms in the water. In practice, this is achieved by heating the water at 60℃-85℃ for a specific time (e.g., 30 seconds to 15 minutes), thereby killing pathogenic bacteria (such as E. coli, Salmonella, Legionella, etc.) and the vast majority of putrefactive bacteria. Furthermore, this temperature does not decompose most nutrients, preserving small molecules such as lactose and protein, providing a favorable environment for the fermentation and growth of the complex microorganisms.

[0072] The methods not described in detail in this embodiment are the same as in Embodiment 2. The lactose measurement method in this embodiment is consistent with step (2) of the simulated lactose residual concentration in water in Embodiment 3, and the protein measurement method is consistent with step (2) of the simulated protein residual concentration in water in Embodiment 3. The results are shown in Table 1. The compound bacteria (ART-27 / AF 7-2) had a residual lactose content of 0.85 g / L in pasteurized water after 48 h, with a fermentation utilization rate of 81.76%. This is 25.65% higher than single-strain lactic acid bacteria fermentation and 15.13% higher than single-strain yeast fermentation. The compound bacteria (ART-27 / AF 7-2) had a residual protein content of 0.57 g / L in pasteurized water after 48 h, with a fermentation utilization rate of 88.44%. This is 12.98% higher than single-strain lactic acid bacteria fermentation and 14.4% higher than single-strain yeast fermentation. It can also be seen that the Kluyveromyces martensii ART-27 and Lactobacillus fermentum AF 7-2 in the compound of the present invention have a synergistic effect, which can synergistically improve the utilization rate of lactose fermentation and protein fermentation in the rinse water treated by pasteurization.

[0073] Table 1. Growth of the compound bacteria using actual rinse water and consumption of dairy components.

[0074] Example 5: Water quality parameters after actual water treatment (1) Lactic acid bacteria AF 7-2 and yeast ART-27 were inoculated at a rate of 2% (AF 7-2 and ART-27 each accounted for 1% of the inoculation rate) and statically cultured in pasteurized water (obtained from a dairy factory) at 30-32℃ for 48 h. The fermented water sample was taken and diluted 10 times with 10×PBS for analysis.

[0075] (2) COD determination: 6.3 g of potassium dichromate powder was added to the sample. In a strongly acidic solution, a special reagent was used as an oxidant and catalyst. After the sample was digested at 165℃ for 10 min, the potassium dichromate was reduced to trivalent chromium by organic matter in the water. The content of trivalent chromium ions was determined at a specific wavelength of 600 nm. The COD concentration was determined by colorimetric method. Open the digester and add 3 ml of distilled water to a sealed test tube as a blank. Take 3 mL of the same sample from each test tube corresponding to its number. Add 1 ml of reagent C1 (90 mL water, 10 mL H2SO4, 6.3 g potassium dichromate) to each tube, and then add reagent C2 (500 mL concentrated sulfuric acid, 7.3 g silver sulfate) to each tube. Shake well and place the tubes in the digestion wells of zone A for 10 min. After digestion, carefully remove the tubes and place them on a test tube rack to air cool for 2 minutes. After cooling, open the caps and add 3 mL of distilled water to each tube. Close the caps, shake well, and place the tubes in the test tube rack to cool to room temperature in water. After cooling, pour the solution from the tubes into a 3 cm cuvette and place them in a rapid analyzer to read the COD value under the COD curve. The results are shown in Table 2. The COD of the untreated dairy wastewater was 5697.30 mg / L. The COD value of the water sample after fermentation was 1240.03 mg / L, and the removal rate reached 78.24%. After fermentation treatment with lactic acid bacteria and yeast, the COD removal efficiency of the experimental group was significantly improved.

[0076] (3) TN determination: Turn on the digester, and start heating the total phosphorus and total nitrogen instruments. Add 5 ml of distilled water to the sealed test tube as a blank. Take the remaining test tubes according to their numbers corresponding to the water samples being tested, with the sample volume being 5 ml each. Add 2.5 mL of total nitrogen reagent 1 (10.5 g potassium persulfate, 4.05 g sodium hydroxide, and 250 mL of distilled water) to each test tube. After tightening the cap and shaking well, carefully place the test tube into the digestion well in area B and digest at 121℃ for 30 min. After digestion, place all the test tubes into the test tube rack and cool for 2 min. After cooling, add 5 mL of total nitrogen reagent 2 (1% hydrochloric acid) to each test tube, tighten the cap and shake well, let stand for 10 minutes, and pour into a 1 cm quartz cuvette. Read the colorimetric value under the total nitrogen curve. The results are shown in Table 2. The TN of the untreated dairy wastewater was 327.31 mg / L. After treatment with microbial fermentation, the total nitrogen (TN) in the experimental group was 63.28 mg / L, indicating a significant improvement in TN removal efficiency. This result demonstrates a significant degradation effect on nitrogen-containing organic matter in the target wastewater.

[0077] (4) SS Suspended Solids Determination: Using flat-eyed toothless tweezers, pick up the microporous filter membrane (CN-CA filter membrane, pore size 0.45 μm, diameter 60 mm) and place it in a pre-weighed weighing bottle. Transfer it to an oven and dry it at 103-105℃ for half an hour. Remove it, cool it, and weigh it until the weight difference between two weighings is ≤0.2 mg. Place the pre-weighed microporous filter membrane correctly in the filter membrane filter, wet the filter membrane with distilled water, and continuously aspirate. The sample obtained in step (1) of Example 5 is filtered by 100 mL aspiration. Make sure all the water passes through the filter membrane. Wash it three times with 10 mL of distilled water each time, and continue aspiration to remove trace water. After stopping aspiration, carefully remove the filter membrane carrying the suspended solids and place it in the original pre-weighed weighing bottle. Transfer it to an oven and dry it at 105℃ for 1 h. Transfer it to a desiccator and cool it to room temperature. Weigh it. Repeat drying, cooling, and weighing until the weight difference between two weighings is ≤0.4 mg. The results are shown in Table 2. The suspended solids (SS) in the untreated dairy wastewater was 37.28 mg / L. After treatment with microbial fermentation, the suspended solids (SS) in the experimental group was 6.00 mg / L, which met the prescribed emission limit (<10 mg / L). The effect after fermentation is shown in the following figure. Figure 5 As shown.

[0078] Table 2. Determination of discharge indicators for fermentation rinse water after 48 hours.

[0079] Example 6 The seed culture obtained in step (1) of Example 2 was inoculated into 100 mL of simulated water at an inoculation rate of 2% (Lactobacillus fermentum AF7-2 and Kluyveromyces martensii ART-27, with AF7-2 and ART-27 each accounting for 1% of the inoculation rate). Fermentation was carried out for 48 h to obtain the fermentation broth (lactose 5 g / L, protein 5 g / L, manganese sulfate 0.02 g / L, magnesium sulfate 0.5 g / L, dipotassium hydrogen phosphate 2 g / L, solvent: water). The broth was transferred into a 200 mL centrifuge cup and centrifuged at 5000 r / min for 10 min. After removing the supernatant, the precipitate was recovered. The precipitate was freeze-dried under vacuum for 12 h to constant weight, and its dry weight was measured.

[0080] The Kjeldahl method was used to determine the protein content of the bacterial cells. After collecting the lyophilized cells to constant weight, 0.5 g of the constant-dry sample was weighed, and 2 tablets of Kjeltabs Cu3, 5 tablets, 2 tablets of defoamer, and 12 mL of concentrated sulfuric acid were added to the digestion tube. The cells were digested in a graphite digester at 240 ℃ for 30 min, then at 420 ℃ for 1 h. After cooling, the digestion tube was placed in an automated Kjeldahl nitrogen analyzer to measure the protein content (%). The protein yield was calculated using the formula: Protein yield (g / L) = Dry weight of bacterial cells (g / L) × Protein content (%). The OD values ​​of lactic acid bacteria, yeast, and mixed bacteria were also determined. 600 The results, including bacterial dry weight (g / L), protein content (%), and bacterial protein content (g / L), were measured. Table 3 shows that the bacterial protein content of the compound bacteria was 0.63 g / L, representing increases of 38.10% and 69.84% compared to the control group, respectively. Through simulated water fermentation, it was demonstrated that in the process of recycling water resources, organic components such as whey protein can be converted into high-value-added bacterial protein through microbial fermentation. This bacterial protein can be used as feed protein, a nutritional fortifier, or a raw material for further processing, realizing the resource utilization and high-value utilization of waste materials. It can also be seen that *Kluyveromyces martensii* ART-27 and *Lactobacillus fermentum* AF 7-2 in the compound agent of this invention have a synergistic effect, which can synergistically improve the conversion of organic components such as whey protein into high-value-added bacterial protein through microbial fermentation in dairy rinsing water.

[0081] Table 3. Protein content of different bacterial species

[0082] Although embodiments of the invention have been disclosed for illustrative purposes, those skilled in the art will understand that various substitutions, variations, and modifications are possible without departing from the spirit and scope of the invention and the appended claims. Therefore, the scope of the invention is not limited to the contents disclosed in the embodiments.

Claims

1. A compound microbial agent for treating dairy product rinsing water, characterized in that: The compound microbial agent includes *Max Kluyveromyces* (…). Kluyveromyces marxianus ART-27 and Lactobacillus fermentum ( Lactobacillus fermentum ); The taxonomic name for *Kluyveromyces martensii* ART-27 is: *Kluyveromyces martensii* ( Kluyveromyces marxianus The accession number is CGMCC No.39119, the accession date is January 7, 2026, and the depositary institution is the China General Microbiological Culture Collection Center (CGMCC), No.3, No.1 Beichen West Road, Chaoyang District, Beijing.

2. The compound microbial agent according to claim 1, characterized in that: The fermented lactobacillus includes Lactobacillus AF7-2, Lactobacillus AF1-3, Lactobacillus AF1-5, Lactobacillus ATCC 9338, and Lactobacillus CECT5716.

3. The compound microbial agent according to claim 1 or 2, characterized in that, The volume ratio of *Lactobacillus fermentum* to *Kluyveromyces masculinus* ART-27 is 1:0.5-1:

3.

4. The application of the compound microbial agent as described in any one of claims 1 to 3 in the treatment of low-concentration dairy water.

5. The application according to claim 4, characterized in that: The compound bacteria can utilize low-concentration dairy products to rinse away lactose and protein in the water through fermentation. Alternatively, the dairy water may include dairy wastewater, dairy pipeline flushing water, dairy tank flushing water, whey, etc.

6. The method for preparing the compound microbial agent according to any one of claims 1 to 3, characterized in that: Includes the following steps: (1) Prepare Lactobacillus fermentation seed culture and Kluyveromyces ART-27 seed culture respectively; (2) Wash the two seed cultures from step (1) with 10×PBS, repeat three times, and then adjust to OD. 600 A bacterial suspension with a value of 1; (3) The two bacterial suspensions obtained in step (2) are mixed in a volume ratio of 1:0.5-1:3 to prepare a compound bacterial suspension, thus obtaining a compound bacterial agent.

7. The preparation method according to claim 6, characterized in that: In step (1), the method for preparing the fermented Lactobacillus seed liquid is as follows: the strain is streaked to activate it, a single colony is picked and inoculated into MRS liquid medium, and then incubated at 37°C for 12-14 hours.

8. The preparation method according to claim 6 or 7, characterized in that: In step (1), the method for preparing the Kluyveromyces ART-27 seed culture is as follows: the Kluyveromyces ART-27 strain is activated by streaking, a single colony is picked and inoculated into YPD liquid medium, and then incubated at 30°C for 16-18 h. Alternatively, in step (3), the bacteria are mixed in a volume ratio of 1:1 to prepare a compound bacterial suspension.

9. A method for treating low-concentration dairy product rinsing water using the compound microbial agent according to any one of claims 1 to 3, characterized in that: Includes the following steps: The compound microbial agent is inoculated into the low-concentration dairy product rinsing water to be treated for fermentation.

10. The method according to claim 9, characterized in that: The total inoculation amount of the compound microbial agent is 0.5-7% of the volume of the low-concentration dairy product rinsing water.

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