A ferment for improving the quality of fermented sweet corn juice and a method for preparing fermented sweet corn juice

Abstract

This invention belongs to the field of microbial technology, specifically relating to a starter culture for improving the quality of fermented sweet corn juice and a method for preparing fermented sweet corn juice. The starter culture is a compound of *Lactobacillus plantarum* (CGMCC No. 3151) and *Bifidobacterium longum* subsp. *infantii* (CCTCC NO: M20221253). The starter culture is inoculated into a mixture of saccharified sweet corn juice and hydrolyzed rice protein, fermented for 3-5 hours, and then pasteurized to obtain fermented sweet corn juice. The resulting fermented sweet corn juice exhibits suitable levels of total sugar, pH, total acidity, reducing sugar content, and precipitation rate, significantly increased total phenolic content, strong DPPH free radical scavenging ability, and harmonious flavor, making it suitable for industrial application.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a fermenting agent for improving the quality of fermented sweet corn juice and a method for preparing fermented sweet corn juice. Background Technology

[0002] Sweet corn (Zea mays L. var. saccharata) is a special variant of maize, and due to its unique flavor, rich nutrition, and significant economic benefits, it has become an important crop of global interest. Sweet corn juice, made from sweet corn, is highly nutritious and flavorful, making it popular with consumers. However, sweet corn juice is prone to problems such as starch sedimentation, monotonous flavor, and unstable quality during storage, which hinders its industrial development.

[0003] Lactic acid bacteria fermentation technology is widely recognized as an effective means of improving the quality of fruit, vegetable, and grain beverages because it can impart active probiotics, unique flavors, and nutritional properties to food. Through microbial metabolic activities, fermentation can give products a unique lactic acid flavor, enrich their taste, and extend their shelf life; it can also degrade anti-nutritional components and synthesize functional active substances, thereby enhancing the health value of the product. Among the many lactic acid bacteria, *Lactobacillus plantarum* (Lactobacillus plantarum) is particularly beneficial. Lactobacillus plantarum Due to their strong environmental adaptability, acid-producing characteristics, and potential probiotic functions, *Lactobacillus plantarum* is often used as a core strain in fermentation engineering. While traditional single-strain fermentation offers controllable processes, its narrow metabolic spectrum may lead to insufficient flavor complexity or limited functional enhancement in the product. For example, CN118648655A provides a sweet corn fermented beverage that uses *Lactobacillus plantarum*, *Lactobacillus gasseri*, and *Lactobacillus reuteri* as starter cultures, but its total phenol content is less than 10 μg / mL, thus limiting its antioxidant capacity. Dong Yangyang et al. investigated the effects of *Lactobacillus reuteri*, *Lactobacillus gasseri*, *Lactobacillus plantarum*, and *Lactobacillus mucilaginosus* on the nutrition and quality of sweet corn, finding significant differences in the utilization rates of total sugar, reducing sugar, and protein among different strains. Except for *Lactobacillus plantarum*, the other three strains did not alter the flavor composition of the corn juice, indicating that the influence of different strains on sweet corn juice fermentation is quite complex.

[0004] Currently, there is a lack of in-depth research and comparison regarding the fermentation characteristics, metabolic interactions, and systematic improvement effects on the overall quality of different microbial combinations in sweet corn juice substrates. This limits the development and utilization of compound fermentation agents in sweet corn juice fermentation products. Further in-depth research is needed on how to improve the nutritional value, stability, content of beneficial substances, flavor, and sensory evaluation of fermented sweet corn juice. Summary of the Invention

[0005] To address the above problems, the purpose of this invention is to provide a starter culture for improving the quality of fermented sweet corn juice and a method for preparing fermented sweet corn juice. The technical solution of this invention is as follows: A starter culture for improving the quality of fermented sweet corn juice, wherein the starter culture uses *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infanthi*, wherein the *Lactobacillus plantarum* strain has the preservation number CGMCC No. 3151; and the *Bifidobacterium longum* subsp. *infanthi* strain has the preservation number CCTCC NO: M20221253. Preferably, the viable count ratio of *Lactobacillus plantarum* to *Bifidobacterium longum* subsp. *infanthi* is 1:(1~3).

[0006] A compound microbial agent is disclosed for use as a fermentation agent for sweet corn juice. The fermentation strains used in the compound microbial agent are *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infanthum*. The *Lactobacillus plantarum* is *Lactobacillus plantarum* KLDS1.0391 with accession number CGMCC No. 3151; the *Bifidobacterium longum* subsp. *infanthum* is *Bifidobacterium longum* subsp. *infanthum* with accession number CCTCC NO: M20221253. Preferably, the live bacteria ratio of *Lactobacillus plantarum* to *Bifidobacterium longum* subsp. *infanthum* in the compound microbial agent is 1:(1~3).

[0007] A method for preparing fermented sweet corn juice includes the following steps: Preparation of S1 saccharified corn juice: Sweet corn is pulped with water to make initial juice. After pulping, the coarse filtrate is obtained by filtration. α-amylase is added, liquefaction is carried out, and the residue is removed by filtration. After enzyme inactivation, corn juice is obtained by adding saccharifying enzyme for saccharification. Enzyme inactivation is carried out again to obtain saccharified corn juice. Preparation of S2 rice hydrolyzed protein mixed with saccharified corn juice: slowly add rice hydrolyzed protein to saccharified corn juice at 40℃~65℃, mix well and keep warm for 20min~50min, sterilize, and obtain rice hydrolyzed protein mixed with saccharified corn juice. S3: Corn juice fermentation: Lactobacillus plantarum with preservation number CGMCC No.3151 and Bifidobacterium longum subsp. infantis with preservation number CCTCC NO: M20221253 were co-inoculated into the saccharified corn juice mixed with rice hydrolysate obtained in step S2. After fermentation, the mixture was sterilized to obtain fermented sweet corn juice.

[0008] Preferably, the amount of α-amylase used in step S1 is 0.1% to 0.3% of the weight of the crude filtrate; the amount of saccharifying enzyme used is 0.2% to 0.4% of the weight of the corn juice.

[0009] Preferably, the liquefaction conditions in step S1 are: liquefaction at 50℃~70℃ for 0.8h~1.5h; and the saccharification conditions are: saccharification at 50℃~70℃ for 2h~4h.

[0010] Preferably, in step S2, the amount of rice hydrolyzed protein added is 0.2% to 0.4% of the total weight of the rice hydrolyzed protein mixed with saccharified corn juice.

[0011] Preferably, in step S3, the inoculation amount of *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantii* in the rice hydrolysate mixed with saccharified corn juice is 1:(1~3) based on the live bacteria ratio.

[0012] Preferably, in step S3, the total inoculation amount of *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantii* in the rice hydrolysate mixed with saccharified corn juice is 0.001 × 10⁻⁶. 7 CFU / mL ~1000×10 7 CFU / mL; More preferably, in step S3, the total inoculation amount of *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantitidis* in the rice hydrolysate mixed with saccharified corn juice is 0.1 × 10⁻⁶ CFU / mL. 7 CFU / mL ~15×10 7 CFU / mL.

[0013] Preferably, in step S3, the fermentation conditions for the rice hydrolysate protein mixed with saccharified corn juice are 30℃~40℃ until the pH drops to 4.0~4.5.

[0014] Beneficial effects

[0015] This invention systematically studied the effects of different lactic acid bacteria and bifidobacteria on the fermentation of sweet corn juice by *Lactobacillus plantarum*. It compared the effects of *Lactobacillus plantarum* fermentation alone and combined fermentation with other strains on the physicochemical properties, antioxidant activity, flavor, and sensory quality of sweet corn juice. The results showed that the combined fermentation of sweet corn juice with *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantii* Y46 resulted in a shorter fermentation time (only 3-5 hours), and the product exhibited suitable levels of total sugar content, pH, total acid content, reducing sugar content, and precipitation rate (stability). Furthermore, it had the highest total phenol content, the highest DPPH scavenging rate, and the most outstanding flavor and sensory scores, making it the optimal fermentation agent combination. Attached Figure Description

[0016] Figure 1 Graph showing the total sugar content of sweet corn juice fermented by different bacterial strains; Figure 2 A graph showing the pH test results of sweet corn juice fermented by different bacterial strains; Figure 3 Graph showing the total acidity test results of sweet corn juice fermented by different bacterial strains; Figure 4 The graph shows the results of reducing sugar content detection in sweet corn juice fermented by different bacterial strains; Figure 5 Figure showing the results of total phenol concentration detection in sweet corn juice fermented by different bacterial strains; Figure 6 Figure 1 shows the results of free radical scavenging rate detection for sweet corn juice fermented by different bacterial strains. Figure 7 The graph shows the sedimentation rate of sweet corn juice fermented by different bacterial strains. Figure 8 Radar analysis results of flavor profiles for sweet corn juice fermented with different bacterial strains; Figure 9 The results of PCA analysis of sweet corn juice fermented by different bacterial strains using an electronic nose are shown in the figure. Detailed Implementation

[0017] The technical solution and effects of the present invention will be shown and described below with reference to specific embodiments and accompanying drawings. The embodiments are only for illustrating the technical solution of the present invention and are not considered as limiting the scope of protection. The letters a, b, c, d, e, and d used to indicate significant differences between groups in the accompanying drawings have the following meanings: when these letters are different between groups, it indicates that there is a significant difference between the groups (…). P <0.05).

[0018] The following examples illustrate the sources of materials and instruments used in the specific experimental operations: The sweet corn kernels were quick-frozen fruit sweet corn provided by Daqing Laojieji Food Co., Ltd.; the food-grade rice protein (i.e., hydrolyzed rice protein) was purchased from Xintai Food Chemical Co., Ltd.; the lactic acid bacteria used in the experiment were: *Lactobacillus rhamnosus* ATCC7469, *Lactobacillus paracasei* CICC6105, *Lactobacillus casei* ATCC393, and *Bifidobacterium longum* subsp. *infant* ATCC15697, which were provided by their respective bacterial culture centers; *Lactobacillus plantarum* KLDS1.0391 (CGMCC No. 3151), *Bifidobacterium longum* subsp. *infant* Y46 (CCTCC NO: M20221253), and *Bifidobacterium bifidum* ZLY10 (CCTCC NO: M2023527) were all screened by our laboratory and preserved in glycerol at −80℃, and have been patented by our institution.

[0019] Lactobacillus plantarum KLDS1.0391 has been published in CN101812414B, with the accession number CGMCCNo.3151. It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at the Institute of Microbiology, Chinese Academy of Sciences, No. 1 Beichen West Road, Chaoyang District, Beijing, on June 29, 2009. Its taxonomic name is... Lactobacillus plantarum.

[0020] Bifidobacterium longum subspecies Y46 has been published in CN115354009A, with accession number CCTCC NO: M20221253, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on August 9, 2022. Its taxonomic name is... Bifidobacterium longumsubsp. infantis Named Bifidobacterium longumsubsp. infantis Y46 (hereinafter referred to as Y46).

[0021] Bifidobacterium bifidum ZLY10 has been disclosed in CN118028171A, with accession number CCTCC NO: M2023527, deposited at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, on April 12, 2023. Its taxonomic name is... Bifidobacterium bifidum ZLY10.

[0022] Gallic acid, anhydrous ethanol, DNS chromogenic reagent (3,5-dinitrosalicylic acid), concentrated sulfuric acid, D-anhydrous glucose, phenol, Folin-Ciocalteu, n-hexane, NaOH, and KOH were all purchased from Sinopharm Chemical Reagent Co., Ltd. for analytical grade. MRS medium (20221215) was purchased from Qingdao Haibo Biotechnology Co., Ltd.; Tween 80 (20230401) was purchased from Tianjin Ruijinte Chemical Co., Ltd.; and trichloroacetic acid (20220919) was purchased from Tianjin Zhiyuan Chemical Reagent Co., Ltd.

[0023] The following instruments were purchased: a clean bench (SW-CJ-1FD) from Suzhou Antai Air Technology Co., Ltd.; an H1750R high-speed benchtop refrigerated centrifuge from Hunan Xiangyi Laboratory Instrument Development Co., Ltd.; a GR85DA vertical pressure steam sterilizer from Zhiwei (Xiamen) Instrument Co., Ltd.; a PB-10 benchtop pH meter from Sartorius Scientific Instruments (Beijing) Co., Ltd.; a CMax Plus microplate reader from Meigu Molecular Instruments (Shanghai) Co., Ltd.; an electric thermostatic incubator (DHP-9162) from Shanghai Yiheng Scientific Instruments Co., Ltd.; an AE100S electronic analytical balance from Mettler Toledo Instruments GmbH, Switzerland; a Q15 soymilk maker from Joyoung Co., Ltd.; a BM-04 digital display saccharimeter from Tianjin Liaowang Optoelectronic Technology Co., Ltd.; a ZE6000 colorimeter from Denshoku Kogyo Co., Ltd., Japan; and a PEN3 electronic nose from Leica GmbH, Germany.

[0024] Example 1: Preparation of different fermented sweet corn juices

[0025] 1. Preparation of corn juice and saccharified corn juice

[0026] The kernels obtained by shelling sweet corn and removing the cob (corn cob) are washed and mixed with 5 times their weight of drinking water. The mixture is then blended in a soymilk maker to produce initial juice. After blending, the juice is filtered through cheesecloth to obtain a coarse filtrate. The pH of the coarse filtrate is measured after cooling and used as the initial pH. α-amylase (0.2% of the weight of the coarse filtrate) is added, and the mixture is liquefied at 60℃ for 1 hour. The mixture is then filtered through cheesecloth to remove residue, and the enzyme is inactivated at 80℃ for 30 minutes to obtain corn juice. Saccharifying enzyme (0.3% of the weight of the corn juice) is added, and saccharification is performed at 60℃ for 180 minutes. After adjusting the pH back to the initial value, the enzyme is inactivated at 80℃ for 30 minutes to obtain saccharified corn juice.

[0027] 2. Addition of hydrolyzed rice protein

[0028] Slowly add rice hydrolyzed protein to saccharified corn juice at 60℃ (final concentration of rice hydrolyzed protein is 3wt%), mix well, keep warm for 30 minutes, and sterilize at 95~100℃ for 5 minutes to obtain rice hydrolyzed protein mixed with saccharified corn juice.

[0029] 3. Activation and subculturing of strains and preparation of fermentation broth

[0030] After thawing the *Lactobacillus plantarum* KLDS1.0391 (hereinafter referred to as LP), *Lactobacillus rhamnosus* ATCC7469 (hereinafter referred to as LR), *Lactobacillus paracasei* CICC6105 (hereinafter referred to as LPC), *Lactobacillus casei* ATCC393 (hereinafter referred to as LC), *Bifidobacterium longum* subsp. *infantii* Y46 (hereinafter referred to as Y46), *Bifidobacterium longum* subsp. *infantii* ATCC 15697 (hereinafter referred to as 97), and *Bifidobacterium bifidum* ZLY10 (hereinafter referred to as Y10) frozen at -80℃, they were respectively administered at 3~8×10⁻⁶. 6 Inoculate with CFU / mL into MRS liquid medium containing 0.05 wt% cysteine ​​hydrochloride and incubate at 37°C for 24 h. After two consecutive subcultures, collect the cells by centrifugation at 3500g for 5 min, and wash the cells three times with sterile water. Finally, resuspend the cells in sterile water to prepare a fermentation suspension for later use, with a viable cell concentration of 5 × 10⁻⁶. 8 CFU / mL.

[0031] 4. Fermentation of corn juice

[0032] Take 100 mL of sterilized rice hydrolyzed protein mixed with saccharified corn juice, and mix at 1.5 × 10⁻⁶. 7 Different fermentation suspensions were inoculated at an inoculum volume of CFU / mL. When LP was mixed with other strains, the inoculum volume ratio of LP to other strains was 1:1 (i.e., the inoculum volumes of LP and other strains were 7.5 × 10⁻⁶). 6 CFU / mL; cultured in a 37℃ incubator, with dynamic pH monitoring every hour. When the pH dropped below 5.0, monitoring was adjusted to every half hour, and a small sample was taken for sensory evaluation until the pH dropped to 4.2, at which point fermentation sampling was stopped (approximately 3-5 hours). After fermentation, pasteurization was performed at 72℃ for 15 minutes to obtain fermented sweet corn juice.

[0033] Example 2: Detection and evaluation of different fermented sweet corn juices

[0034] This embodiment is a follow-up study of the experiment in Example 1, which involves the detection and evaluation of fermented sweet corn juice prepared by different strains and combinations according to the method in Example 1 (the only difference being that fermentation was stopped after 4 hours and pasteurized in the "corn juice fermentation" step).

[0035] 1. Detection indicators and methods

[0036] 1.1 Physicochemical Indicators

[0037] 1.1.1 Determination of total sugar content

[0038] Determination using a saccharimeter: After calibration with sterile distilled water, use a glass rod to apply the sample to the saccharimeter for measurement. Before each measurement, the saccharimeter should be washed with sterile distilled water and any residual moisture removed with absorbent paper. Each sample should be tested in triplicate, and the final average value should be taken.

[0039] 1.1.2 pH Measurement

[0040] The pH meter was used for measurement: First, the pH meter electrode was rinsed with sterile distilled water, and then residual moisture was removed with absorbent paper. During the measurement, the electrode was kept in the solution for 1-2 minutes, and the data was recorded after the reading stabilized to ensure accuracy. Each sample was measured in triplicate, and the final average value was taken.

[0041] 1.1.3 Determination of total acidity

[0042] According to the pH meter potentiometric titration method in GB12456-2021, weigh 25 mL of fermented sweet corn juice into a beaker, place the beaker on a magnetic stirrer, neutralize the sample solution with NaOH standard solution, and observe the pH change of the solution at any time. Continue until the pH of the solution reaches the endpoint of 8.2, and record the volume of NaOH standard titration solution consumed to calculate the total acid content of the solution.

[0043] 1.1.4 Determination of reducing sugars

[0044] According to the direct titration method in GB5009.7-2016, 80 mL of fermented sweet corn juice was weighed into an Erlenmeyer flask. After removing the protein, methylene blue was used as an indicator to titrate the standardized alkaline copper tartrate solution (which had been standardized with a reducing sugar standard solution) under heating conditions. The reducing sugar content was calculated based on the volume of the sample solution consumed.

[0045] 1.1.5 Determination of total phenols

[0046] The total phenol content was determined using the Folin-Ciocalteu colorimetric method. 1 mL of fermented sweet corn juice was weighed, and 3.5 mL of 70% ethanol solution was placed in a 5 mL brown centrifuge tube. The mixture was then sonicated at 30℃ for 0.5 h, centrifuged at 9000×g for 5 min, and the supernatant was transferred to a 10 mL volumetric flask. The residue was extracted again with 3.5 mL of 70% ethanol solution, and this process was repeated once. The supernatants from both extractions were combined and diluted to volume with 70% ethanol solution. 1.0 mL of the sample was taken, and 5.0 mL of water, 1 mL of Folin-Ciocalteu colorimetric reagent, and 3 mL of 7.5% sodium carbonate solution were added. After vortexing and color development, the mixture was allowed to stand for 2 h. The absorbance of the sample was measured at 765 nm, and a gallic acid standard curve was plotted. The regression equation for the gallic acid standard content determination was calculated as: y = 0.0754x + 0.0681, R0. 2 =0.9992. Where x is the absorbance and y is the concentration of gallic acid (µg / mL).

[0047] 1.1.6 Determination of DPPH free radical scavenging rate

[0048] Prepare a 0.2 mmol / L DPPH powder solution in anhydrous ethanol. Mix the DPPH powder with the sample at a 1:1 volume ratio and react at room temperature in the dark for 30 min. Measure the absorbance at 517 nm. Perform triplicate measurements for each sample and take the average value. The DPPH free radical scavenging rate is calculated using Formula I:

[0049] (Formula I)

[0050] In the formula: A1 represents the absorbance value of the sample solution; A2 represents the absorbance value measured by mixing the sample with anhydrous ethanol; A0 represents the absorbance value measured by mixing anhydrous ethanol with DPPH working solution.

[0051] 1.1.7 Determination of corn juice sedimentation rate

[0052] The prepared fermented sweet corn juice sample was centrifuged at 3000 × g for 20 min at room temperature. After centrifugation, the percentage of the total liquid weight in the precipitate was calculated, and the sedimentation rate was calculated according to Formula II:

[0053] (Formula II)

[0054] 1.2 Electronic Nose Analysis

[0055] Sample pretreatment: Take 10 mL of fermented sweet corn juice sample into a 50 mL headspace vial, seal it, and cap it. After equilibration for 30 min, perform the detection. Each sample is measured 6 times. Detection conditions: Headspace sampling method is used. Sensor performance is shown in Table 1. Electronic nose sample preparation time: 5 s, cleaning time: 80 s, detection time: 80 s. Analyze the flavor components of fermented sweet corn juice by performing 3 replicate experiments. Data from 74 to 76 s are relatively stable and can be analyzed.

[0056] Table 1 Performance of different sensors in the PEN3 electronic nose

[0057] 1.3 Microbial Indicator Detection

[0058] The microbial indicators of fermented sweet corn juice were tested in accordance with the microbial limit requirements in GB7101-2022.

[0059] 1.4 Sensory Evaluation

[0060] The fermented sweet corn juice was tested using a consumer testing method (by 15 professionals from the College of Food Science; the sample information was blinded). The samples were evaluated based on color, flavor, texture and taste, and scored. The scoring criteria are shown in Table 2.

[0061] Table 2 Sensory Evaluation Form

[0062] The above detection and evaluation data were analyzed using SPSS 25 software for univariate statistical analysis and Duncan's multiple comparisons of differences between groups, and plotted using Origin 2024 software. P < 0.05 was considered statistically significant.

[0063] 2 Results and Analysis

[0064] 2.1 Effects of fermentation by different strains on the physicochemical properties of sweet corn juice

[0065] 2.1.1 Total sugar content

[0066] The total sugar content of the final product was measured after fermentation, and the results are as follows: Figure 1As shown, different strains and complex strains had varying effects on the total sugar content of fermented sweet corn juice. The LP+Y10 group had the lowest total sugar content at 8.80% (significantly lower than other groups); the LP, LP+LR, LP+LPC, and LP+Y46 groups had moderate total sugar content with no significant differences among groups (P>0.05). The LP+LC group had the highest total sugar content at 9.3% (significantly higher than other groups). The LP+Y46 group had a moderate total sugar content, neither excessively consuming nor significantly retaining sugar. Its mild sugar metabolism characteristics make it an excellent strain resource for balancing fermentation flavor and natural sweetness, thus showing potential for application in optimizing product palatability.

[0067] 2.1.2 pH value

[0068] After fermentation, the pH of the final product was measured. Significant differences in the acidification capacity of different strains and complex cultures were observed, with the final pH decreasing in all groups after fermentation. Figure 2 As shown, the LP+LC and LP+LR groups exhibited the fastest acidification rates, with a significantly lower final pH compared to other groups. The LP+Y46 and LP-only fermentation groups had moderate pH values, while the LP+LPC and LP+Y10 groups showed the smallest pH decreases, indicating relatively weaker acidification capabilities. LP rapidly consumes fermentable sugars and produces lactic acid in the early stages of fermentation, leading to environmental acidification. If the accompanying bacteria have good acid tolerance, they can continue metabolism under low pH conditions, forming a continuous acid-producing process. In the LP+LC and LP+LR groups, the final pH decreased significantly, indicating a smoother acidification chain in their combination. In contrast, the LP+LPC and LP+Y10 groups had higher pH values, possibly because the accompanying bacteria were inhibited in the acidic environment, preventing the metabolic process from continuing.

[0069] 2.1.3 Total Acidity

[0070] Total acidity reflects the acid-producing capacity of different samples. The total acidity of the final product is measured after fermentation. Figure 3 As shown, the LP+LC group had the highest total acidity (P<0.05), indicating the strongest acid-producing capacity. The total acidity of both the LP+LC and LP+LR groups was significantly higher than that of the LP group, indicating a synergistic effect in acid production between these two groups of bacteria. The total acidity of the LP+Y46 group was lower than that of the LP group, suggesting that this combination exhibited a milder acid-producing intensity. Based on the sensory evaluation results, the LP+Y46 group was the optimal combination, as its total acidity avoided the flavor irritation caused by excessive acidity while ensuring sufficient acidity to inhibit the growth of unwanted microorganisms, thus meeting the balance between quality and flavor requirements for fermented products.

[0071] 2.1.4 Reducing sugar content

[0072] Changes in reducing sugar content can reflect the metabolic levels of carbohydrates in different sample groups. The reducing sugar content of the final product is measured after fermentation. Figure 4 As shown, the LP+LPC group had the highest reducing sugar content, significantly higher than all other groups (P<0.05); while the reducing sugar content of the LP+LC group was significantly lower than that of the LP+LPC and LP+LR groups (P<0.05). Combined with sensory evaluation results, the reducing sugar content of LP+Y46 remained within a moderate range, which is conducive to achieving appropriate metabolism of reducing sugars and avoiding excessive consumption. This characteristic meets the quality requirements of fermented products.

[0073] 2.1.5 Total phenol content

[0074] After fermentation, the total phenol content of the final product was measured. Different fermentation strains significantly affected the total phenol content of the samples, exhibiting a clear strain-dependent effect. For example... Figure 5 As shown, the LP+Y46 group had a higher total phenol content, indicating that the complex bacteria degraded fewer phenolic substances during fermentation and may have promoted the release of some phenolic substances. The differences between samples may be related to the differences in the activity and expression levels of key enzymes carried by different lactic acid bacteria species, thus affecting their ability to metabolize phenolic substances. The total phenol content of the LP+Y46 group was approximately 10.9% higher than that of the LP group.

[0075] To clarify whether Y46 has a specific effect on the total phenol content of fermented sweet corn, the effects of LP combined with Bifidobacterium longum subsp. infantis ATCC 15697 (97) on the total phenol content were further investigated. The results showed that all three could increase the total phenol content of fermented sweet corn juice, but Y46 had the most significant effect.

[0076] 2.1.6 DPPH free radical scavenging rate

[0077] DPPH removal rates showed clear stratification, with significant differences among different bacteria. For example... Figure 6 As shown, the LP+Y46 and LP+Y10 groups exhibited stronger antioxidant capabilities, with the LP+Y46 group showing a significantly higher DPPH scavenging rate than the LP+Y10 group. The LP+LR and LP+LPC groups showed moderate antioxidant capabilities, while the LP+LC group was similar to the LP group, with limited improvement in antioxidant capacity. Fermentation with multiple microorganisms was more effective than single-strain fermentation in enhancing antioxidant capacity.

[0078] 2.1.7 Sedimentation rate

[0079] Sedimentation rate is one of the important indicators reflecting the stability of a sample during fermentation. For example... Figure 7As shown, the precipitation rates of the LP and LP+LR groups were significantly higher than those of the other groups (P<0.05); the precipitation rate of the LP+LC group was significantly lower than that of the other groups (P<0.05); while the precipitation rates of the LP+LPC, LP+Y46, and LP+Y10 groups were in the middle range, with no significant differences among the groups (P>0.05). These results indicate that different compound bacterial fermentations have different effects on sample stability. Except for the LP+LR group, the precipitation rates of the other four groups were significantly lower than those of the LP group (P<0.05), indicating that compound bacterial fermentation is more effective in improving sample stability than LP single fermentation.

[0080] To investigate whether the effect of Y46 on the stability of fermented sweet corn is specific, the effects of Lactobacillus plantarum (LP) and Bifidobacterium longum subsp. infantis ATCC15697 (97) on stability after co-fermentation were further compared. The results showed that 97 could also improve the stability of fermented sweet corn juice, but Y46 had the strongest effect on improving stability.

[0081] 2.2 Effects of different bacterial strains on the flavor of sweet corn juice

[0082] The flavor and electronic nose detection results of the obtained fermented sweet corn products were analyzed using radar analysis and PCA (principal component analysis), respectively. The results are as follows: Figure 8 and Figure 9 As shown.

[0083] The LP+LPC, LP+Y46, and LP+Y10 groups showed high response values ​​on the W1C, W5S, W3C, and W5C sensors, indicating a rich content of volatile flavor compounds, especially aromatics, alcohols, and olefins. This may be due to esters and aldehydes produced during fermentation. In contrast, the LP+LR, LP+LC, and LP groups showed lower response values ​​on most sensors, indicating a relatively simple volatile flavor composition. This may be due to a limited variety of metabolites or the accumulation of certain off-odor components.

[0084] It is worth noting that the LP+LR group showed significantly higher response values ​​in both the W1W and W2W sensors, indicating the possible presence of undesirable sulfide flavors, consistent with the sensory evaluation's description of "off-odor." Overall, the LP+LPC and LP+Y46 groups exhibited superior flavor profiles, possessing typical fermented aromas without any noticeable undesirable odors, which aligns with their high scores in the sensory evaluation.

[0085] 2.3 Effects of multi-strain fermentation on the sensory and microbiological properties of corn juice

[0086] 2.3.1 Microbiological Indicators

[0087] After fermentation, the sweet corn juice was stored at room temperature for 6 months and then subjected to microbial testing. The results showed that the total bacterial count of each group of fermented sweet corn juice was below 100 CFU / mL, and coliform bacteria were not detected. All the above results meet the requirements of GB4789.1-2016 "National Food Safety Standard - General Rules for Microbiological Examination of Food".

[0088] 2.3.2 Sensory evaluation

[0089] The sensory evaluation results of each group of fermented sweet corn juice samples are shown in Table 3. The scores for the same product in Table 3 are the average scores given by 15 professional technicians. The scores of the LP group and the LP+Y10 group were slightly lower, with the differences mainly reflected in texture and flavor. The LP+LR group had the lowest sensory evaluation, indicating that its color, aroma, taste, and texture were all unsatisfactory. Combined with the electronic nose analysis results, this may be due to sulfide substances produced during the complex fermentation process.

[0090] The LP+LPC group and the LP+Y46 group had the same and highest sensory scores, indicating that they were essentially identical in terms of color, aroma, taste, and texture. However, the LP+Y46 group, with its slightly sweet taste, neutralized some of the acidity from normal fermentation, making it more sensorily acceptable. Furthermore, its stability was superior to the LP+LPC group, giving it an advantage in commercial production.

[0091] Table 3 Sensory Evaluation Results

[0092] This invention systematically studied the effects of different lactic acid bacteria and bifidobacteria on the fermentation of sweet corn juice by *Lactobacillus plantarum* (LP). The focus was on comparing the effects of LP fermentation alone versus combined fermentation on the physicochemical properties, antioxidant activity, flavor, and sensory quality of the sweet corn juice. The results showed that the combination of *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantii* Y46 was within the optimal range for total sugar content, pH, total acid content, reducing sugar, and protein content, and exhibited the highest total phenolic content and best stability. This combination also produced the product with the most outstanding sensory scores and was therefore identified as the optimal starter culture combination.

Claims

1. A starter culture for improving the quality of fermented sweet corn juice, characterized in that, The fermentation strains used in the fermentation agent are *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. infantis. The *Lactobacillus plantarum* is the *Lactobacillus plantarum* with the preservation number CGMCC No. 3151; the *Bifidobacterium longum* subsp. infantis is the *Bifidobacterium longum* subsp. infantis with the preservation number CCTCC NO: M20221253.

2. The fermenting agent according to claim 1, characterized in that, The ratio of viable bacteria of *Lactobacillus plantarum* to *Bifidobacterium longum* subsp. *infantii* is 1:(1~3).

3. The use of a compound microbial agent as a fermentation agent for sweet corn juice, characterized in that, The fermentation strains used in the compound microbial agent are *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. infantis; the *Lactobacillus plantarum* is *Lactobacillus plantarum* with accession number CGMCC No. 3151; the *Bifidobacterium longum* subsp. infantis is *Bifidobacterium longum* subsp. infantis with accession number CCTCC NO: M20221253.

4. The use according to claim 3, characterized in that, The ratio of viable Lactobacillus plantarum to Bifidobacterium longum subsp. infantis in the compound microbial agent is 1:(1~3).

5. A method for preparing fermented sweet corn juice, characterized in that, Includes the following steps: Preparation of S1 saccharified corn juice: Sweet corn kernels are mixed with water to make initial juice. After mixing, the juice is filtered to obtain coarse filtrate. α-amylase is added, liquefied, filtered to remove residue, and the enzyme is inactivated to obtain corn juice. Saccharifying enzyme is added to saccharify the juice, and the enzyme is inactivated again to obtain saccharified corn juice. Preparation of S2 rice hydrolyzed protein mixed with saccharified corn juice: Add rice hydrolyzed protein to saccharified corn juice at 40℃~65℃, mix well and keep warm for 20min~50min, then sterilize to obtain rice hydrolyzed protein mixed with saccharified corn juice. S3: Corn juice fermentation: Lactobacillus plantarum with preservation number CGMCC No.3151 and Bifidobacterium longum subsp. infantis with preservation number CCTCCNO: M20221253 were co-inoculated into the saccharified corn juice mixed with rice hydrolysate obtained in step S2. After fermentation, the mixture was sterilized to obtain fermented sweet corn juice.

6. The method for preparing fermented sweet corn juice according to claim 5, characterized in that, In step S1, the amount of α-amylase used is 0.1% to 0.3% of the weight of the crude filtrate; the amount of saccharifying enzyme used is 0.2% to 0.4% of the weight of the corn juice.

7. The method for preparing fermented sweet corn juice according to claim 5, characterized in that, The liquefaction conditions in step S1 are: liquefaction at 50℃~70℃ for 0.8h~1.5h; the saccharification conditions are: saccharification at 50℃~70℃ for 2h~4h.

8. The method for preparing fermented sweet corn juice according to claim 5, characterized in that, In step S2, the amount of rice hydrolyzed protein added is 0.2% to 0.4% of the total weight of the rice hydrolyzed protein mixed with saccharified corn juice.

9. A method for preparing fermented sweet corn juice according to claim 5, characterized in that, In step S3, the inoculation amount of *Lactobacillus plantarum* and *Bifidobacterium longum* subsp. *infantii* in the rice hydrolysate mixed with saccharified corn juice is 1:(1~3) based on the live bacteria ratio.

10. A method for preparing fermented sweet corn juice according to claim 5, characterized in that, In step S3, the fermentation conditions for the rice hydrolysate protein mixed with saccharified corn juice are 30℃~40℃, and fermentation continues until the pH drops to 4.0~4.5.

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