A phenolic acid-rich black sweet corn plant-based yogurt and a preparation method and application thereof

Abstract

This invention discloses a plant-based yogurt rich in phenolic acids from black sweet corn, its preparation method, and its applications. Using black sweet corn juice and soybean flour as a matrix, this invention incorporates *Lactobacillus plantarum* strain BFS1243 into a conventional plant-based yogurt fermentation agent for compound fermentation. The resulting black sweet corn yogurt is a bright pink color, has a delicate texture, low beany odor, and significantly enhanced antioxidant activity. The compound fermentation using strain BFS1243 can cleave the glycosidic bonds on anthocyanin molecules in black sweet corn, directionally degrading and converting large anthocyanin molecules with low absorption rates into small phenolic acids. This solves the problem of easy degradation and low bioavailability of natural anthocyanins in black sweet corn during fermentation and storage. It also shortens fermentation time, improves fermentation efficiency, and significantly improves the flavor and quality of the fermented yogurt, giving it a unique corn aroma and pink appearance, as well as more active substances and better antioxidant activity.

Description

Technical Field

[0001] This invention relates to the field of microbial fermentation and yogurt preparation technology, and more specifically, to a black sweet corn plant-based yogurt rich in phenolic acids, its preparation method, and its application. Background Technology

[0002] With the growing popularity of healthy eating, plant-based yogurt has become increasingly popular due to its lactose-free, low-fat, and fiber-rich properties. However, most commercially available plant-based yogurts are based on grains or soybeans, resulting in significant product homogenization and common defects such as a strong beany taste, coarse texture, and poor gel stability. For example, existing plant-based (soybean-coconut powder) yogurts still suffer from poor stability, a strong beany taste, and poor mouthfeel and flavor.

[0003] Black sweet corn, a novel type of naturally colored fresh corn, is not only rich in soluble sugars and high-quality plant protein, but also contains a large amount of anthocyanins, phenolic acids, and other phytochemicals, exhibiting significant antioxidant and glucose-lipid metabolism-regulating bioactivities. Developing it into plant-based yogurt can not only solve the problem of fresh corn's poor storage life, but also utilize its natural pigments and flavors to mask the beany taste of plant-based bases (such as soybeans).

[0004] However, due to the large molecular weight of natural anthocyanins in black sweet corn, they are not easily absorbed by the human body through the intestinal wall barrier; moreover, they are easily degraded and faded during yogurt processing and storage, resulting in low levels of their active ingredients. Using ordinary commercial starter cultures (such as plant-based yogurt starter culture No. 1) for fermentation is insufficient to effectively convert these functional components, resulting in low levels of anthocyanins, phenolic acids, and other active ingredients in the fermented yogurt. Therefore, improving the content of active substances and flavor of existing plant-based yogurts, converting the large molecular bound functional components in black sweet corn into easily absorbed, highly active small molecules, and solving the problem of poor stability of compounded plant protein gels are key technical challenges that urgently need to be addressed in the development of high-quality black sweet corn yogurt. Therefore, this invention application is proposed. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing black sweet corn fermentation preparation. The present invention provides a black sweet corn plant-based yogurt rich in phenolic acids, its preparation method and application.

[0006] The first objective of this invention is to provide a method for preparing black sweet corn plant-based yogurt rich in phenolic acids.

[0007] A second objective of this invention is to provide a black sweet corn plant-based yogurt rich in phenolic acids.

[0008] The third objective of this invention is to provide *Lactobacillus plantarum* (… Lactobacillus plantarum New applications of strain BFS1243.

[0009] The above-mentioned objective of this invention is achieved through the following technical solution: This invention provides a method for preparing black sweet corn plant-based yogurt rich in phenolic acids, comprising the following steps: S1: Mix black sweet corn juice, soybean flour, and white sugar with water, homogenize to obtain a plant-based emulsion, sterilize and set aside; the mass ratio of black sweet corn juice: soybean flour: white sugar: water is (30~70):(5~10):(5~10):(10~60). S2: A compound fermentation agent is added to the sterilized plant-based emulsion, and fermentation is carried out under constant temperature conditions to obtain fermented curd; the compound fermentation agent contains plant-based yogurt starter No. 1 powder and Lactobacillus plantarum (… Lactobacillus plantarum BFS1243 strain, with a mass ratio of (1~5):1; S3: After fermentation, the curd is refrigerated and then matured to obtain black sweet corn plant-based yogurt rich in phenolic acids.

[0010] This invention uses black sweet corn juice and soybean flour as a base, and combines Lactobacillus plantarum (Lactobacillus) with a conventional yogurt starter culture (plant-based yogurt starter culture No. 1). Lactobacillus plantarum The BFS1243 strain, used in compound fermentation, significantly improves the problems of easily degraded and poorly absorbed natural anthocyanins and low content of active ingredients in fermented black sweet corn. The resulting black sweet corn yogurt is bright pink, has a delicate texture, and low beany odor. Its antioxidant activity is significantly enhanced, with significantly increased DPPH free radical scavenging rate, FRAP iron reducing power, ·OH hydroxyl scavenging rate, and total phenolic content. Its effect is superior to black sweet corn yogurt fermented alone using conventional starter cultures or BFS1243 strain alone. This invention, based on conventional fermentation agents, further combines Lactobacillus plantarum BFS1243 compound fermentation, which can cleave the glycosidic bonds on anthocyanin molecules in black sweet corn, directionally degrading large-molecule anthocyanins with low absorption rates and converting them into small-molecule phenolic acids, such as p-hydroxybenzoic acid and p-coumaric acid. Compared to the original large-molecule anthocyanins, small-molecule phenolic acids have a stronger and more efficient ability to scavenge free radicals and have antioxidant activity in the yogurt system. This greatly improves the permeability and bioavailability of the active substance in the human intestine, further enhancing the activity effect of fermented yogurt. It also significantly solves the problem of easy degradation and low bioavailability of natural anthocyanins in black sweet corn during fermentation and storage.

[0011] Preferably, the black sweet corn is "Yuetian Black Gem No. 1".

[0012] More preferably, fresh black sweet corn is selected, threshed and washed, and then water is added at a mass-volume ratio of 1:1 to make a pulp. After filtration, black sweet corn juice is obtained.

[0013] Preferably, the viable count of plant-based yogurt starter culture No. 1 in S2 is not less than 1.0 × 10⁻⁶. 9 CFU / g, the total viable count of strain BFS1243 is not less than 1.0 × 10⁻⁶. 9 CFU / g.

[0014] In the method provided by this invention, by adding a stabilizer to the raw materials, the problem of poor stability of compound plant protein gel can be further solved, and the stability of yogurt can be significantly improved, thus extending the shelf life of the product.

[0015] Preferably, a stabilizer is also added to the raw materials of S1, and the mass ratio of the black sweet corn juice to the stabilizer is (400~500):(1~3).

[0016] More preferably, the stabilizer is selected from carrageenan, soluble soybean polysaccharide, low-ester pectin, guar gum, or xanthan gum.

[0017] More preferably, the mass ratio of the black sweet corn juice to the stabilizer is 460:1.

[0018] More preferably, the stabilizer is a soluble soybean polysaccharide.

[0019] Furthermore, in S1, based on the total mass of the plant-based emulsion, the amounts of each ingredient added are as follows: soybean flour 5%~9%, white sugar 6.5%~7.5%, black sweet corn juice (based on solid content) 2.8%~6.8%, and temperature stabilizer 0.01%~0.3%.

[0020] Preferably, the plant-based yogurt starter culture No. 1 in S1 contains *Lactococcus lactis* subsp. *lactococcus*, *Lactobacillus casei*, and *Streptococcus thermophilus* subsp. *salivarius*, with a total viable count of not less than 1 × 10⁻⁶. 9 CFU / g.

[0021] Preferably, the sterilization conditions in S1 are: water bath sterilization at 60~90℃ for 10~20 minutes.

[0022] Preferably, the fermentation temperature in S1 is 25~35℃, and the fermentation time is 8~18 hours.

[0023] Preferably, the conditions for post-ripening in S3 are: 12-24 hours of post-ripening at 0-5℃.

[0024] As a preferred embodiment, the present invention provides a more specific preparation method: S1: Select fresh black sweet corn, remove the kernels, wash it, add water at a mass-volume ratio of 1:1 for corn to water, blend it into a pulp, filter it to obtain black sweet corn juice; S2: Add soybean flour, white sugar, stabilizer, and the black sweet corn juice obtained in step S1 to drinking water, make up the volume and mix well, then homogenize to obtain a plant-based emulsion; based on the total mass of the plant-based emulsion, the addition amounts of each raw material are as follows: soybean flour 5%~9%, white sugar 6.5%~7.5%, black sweet corn juice (based on solids content) 2.8%~6.8%, and thickener 0.01%~0.3%; S3: Pasteurize the plant-based emulsion obtained in step S2 and cool it for later use; S4: A compound starter culture is added to the sterilized and cooled plant-based emulsion, and fermentation is carried out under constant temperature conditions to obtain fermented curd; the compound starter culture contains plant-based yogurt starter culture No. 1 powder and Lactobacillus plantarum BFS1243 strain, with a mass ratio of (1~3):1; preferably, Lactobacillus plantarum BFS1243 powder is used, wherein the total viable count of plant-based yogurt starter culture No. 1 powder is not less than 1.0×10⁻⁶. 9 The total viable count of Lactobacillus plantarum BFS1243 bacterial powder is not less than 1.0 × 10⁻⁶ CFU / g. 9 CFU / g; S5: After fermentation, the curd is placed under refrigeration for post-ripening to obtain black sweet corn plant-based yogurt rich in phenolic acids.

[0025] Preferably, the preparation method of the Lactobacillus plantarum BFS1243 bacterial powder is as follows: take 1g of bacterial mud and mix it thoroughly with 5mL of protective agent (the protective agent is a 5% maltodextrin solution), pre-freeze it at 4℃ for 1h, then transfer it to -20℃ for 1h, then transfer it to -80℃ for 12h, and finally transfer it to a freeze dryer at -80℃ for 24h. The obtained bacterial powder is sealed and stored at -20℃.

[0026] More preferably, the total inoculation amount in the black sweet corn juice reaches 0.02 g / L, and the number of viable bacteria per milliliter of the raw material after inoculation is not less than 2 × 10⁻⁶. 6 CFU / mL.

[0027] This invention provides a method that, based on conventional plant-based fermentation agents, incorporates *Lactobacillus plantarum* BFS1243 to promote the degradation of macromolecular anthocyanins in black sweet corn into small-molecule phenolic acids such as p-hydroxybenzoic acid and p-coumaric acid. This significantly increases the total phenolic content and antioxidant capacity of yogurt, solving the problems of insufficient antioxidant activity and low bioavailability of macromolecular anthocyanins in existing plant-based yogurts. Simultaneously, by utilizing *Lactobacillus plantarum* BFS1243 and the plant-based yogurt starter, the excellent synergistic effect of acid production and thickening in a corn-soybean substrate is achieved, significantly shortening the coagulation time by 8 hours compared to fermentation using traditional strains alone. This significantly reduces production time and costs and improves the fermentation efficiency of plant-based yogurt. Using black sweet corn juice as the core base ingredient, this invention not only imparts a unique corn aroma and natural pink color to the yogurt, effectively masking the beany taste, but also, through the synergistic fermentation of the compound strains, perfectly neutralizes the abrupt acidity problem caused by BFS1243 fermentation alone, resulting in a more stable product color and a smoother, more delicate taste.

[0028] The present invention also provides a black sweet corn plant-based yogurt rich in phenolic acids prepared by the above method. The prepared black sweet corn plant-based yogurt is rich in small molecule phenolic acids such as p-hydroxybenzoic acid and p-coumaric acid, and has a bright pink appearance and a unique corn aroma, as well as high nutritional value and antioxidant activity.

[0029] Therefore, the present invention also provides *Lactobacillus plantarum* (… Lactobacillus plantarum The following are new applications of strain BFS1243: Prepare the above-mentioned black sweet corn plant-based yogurt; Promotes the conversion of large anthocyanin molecules in black sweet corn into small phenolic acids; Enhance the antioxidant activity of black sweet corn plant-based yogurt; Increase the total polyphenol content of black sweet corn plant-based yogurt; Increase the content of small-molecule phenolic acids in black sweet corn; Preparation of a formulation to increase the total polyphenols in black sweet corn plant-based yogurt.

[0030] Furthermore, the small molecule phenolic acid is a free phenolic acid with a small molecular weight; specifically, it is a phenolic acid such as p-hydroxybenzoic acid, p-coumaric acid, and trans-cinnamic acid.

[0031] Furthermore, the BFS1243 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 5, 2022, with accession number GDMCC No:62947.

[0032] The present invention has the following beneficial effects: This invention provides a method for preparing plant-based yogurt rich in phenolic acids from black sweet corn. The method uses black sweet corn juice and soybean flour as a matrix, and employs a specific compound fermentation agent (containing conventional plant-based fermentation agents and *Lactobacillus plantarum* BFS1243) for compound fermentation. The resulting plant-based yogurt from black sweet corn is rich in small-molecule active ingredients such as phenolic acids, solving the problems of easy degradation and low bioavailability of anthocyanins in black sweet corn. It also has the following advantages: (1) Enhanced antioxidant activity: The method of this invention uses compound Lactobacillus plantarum BFS1243 for fermentation, which can promote the decomposition of macromolecular anthocyanins in black sweet corn and convert them into small molecule phenolic acid substances such as p-hydroxybenzoic acid and p-coumaric acid; the total phenol content of yogurt after fermentation increases significantly, and the antioxidant activity is significantly increased compared with conventional plant-based fermentation agents.

[0033] (2) Improved fermentation efficiency: The compound fermentation agent containing BFS1243 bacterial powder showed good acid production and thickening performance in corn-soybean substrate, which shortened the coagulation time by 8 hours compared with the original strain alone, effectively shortening the fermentation time and enhancing the acid production capacity.

[0034] (3) Sensory quality improvement: Based on the existing plant-based (soybean-coconut powder) yogurt, using black sweet corn juice instead of the original coconut powder significantly improves the flavor quality of the fermented yogurt, giving it a unique corn aroma and pink appearance. The resulting yogurt is a bright and stable pink color, with a delicate texture, effectively masking the beany smell and possessing a unique corn fragrance. It also has more active substances and better antioxidant activity. Furthermore, the addition of stabilizers further solves the problem of poor stability of the compound plant protein gel, significantly extending the product's shelf life. Attached Figure Description

[0035] Figure 1 The antioxidant activity and antioxidant activity levels of different fermentation groups after 18 hours of fermentation are shown in the figure (CK refers to the fermentation group using conventional fermentation agents; 1:1 refers to the fermentation group using compound agents; BFS1243 refers to the fermentation group using Lactobacillus plantarum BFS1243 alone, the same below).

[0036] Figure 2 The pH values ​​of different fermentation groups after 18 hours of fermentation are shown.

[0037] Figure 3 The viscosity of different fermentation groups after 18 hours of fermentation.

[0038] Figure 4 Sensory quality of different fermentation groups after 18 hours of fermentation.

[0039] Figure 5 The anthocyanin content of different fermentation groups (groups A and B only) was compared with that at the beginning after 18 hours of fermentation.

[0040] Figure 6 Correlation analysis of the changes in the content of various substances during fermentation in different fermentation groups (groups A and B only).

[0041] Figure 7 The effects of different stabilizers at addition levels of 0.1% and 0.3% on the texture of black sweet corn yogurt were investigated.

[0042] Figure 8 The effect of different amounts of added soybean polysaccharides on the water-holding capacity of black sweet corn yogurt during storage.

[0043] Figure 9 The effect of different amounts of added soybean polysaccharides on the viscosity of black sweet corn yogurt during storage.

[0044] Figure 10 The effect of different amounts of added soybean polysaccharides on the sensory quality of black sweet corn yogurt. Detailed Implementation

[0045] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the embodiments do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in this technical field.

[0046] Unless otherwise specified, all reagents and materials used in the following examples are commercially available.

[0047] The conventional plant-based starter culture used in this example is a publicly available yogurt starter culture product: Plant-based yogurt starter culture powder No. 1 purchased from Guangzhou Miaodou Biotechnology Co., Ltd. (Product name: Suju Plant-based Yogurt Starter Culture YOGHURTSTARTER, Production License No.: SC106440112012410, Product Standard Code: QB / T 4575, Viable count ≥1×10⁻⁶). 9 The main components of the compound strain (CFU / g) include Lactococcus lactis subsp., Lactobacillus casei, and Streptococcus thermophilus subsp. (the information on the compound strains is recorded in Chinese Patent Publication No. CN110129243A).

[0048] The *Lactobacillus plantarum* strain BFS1243 used was deposited on November 5, 2022, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No:62947, located at 5th Floor, Building 59, No. 100, Xianlie Middle Road, Guangzhou, China, and is recorded in Chinese patent document CN116875504A.

[0049] Example 1: A preparation process for black sweet corn yogurt with high antioxidant activity (1) Pretreatment: Select fresh “Yuetian Black Gem No. 1” corn, remove the skin and silk, thresh the kernels, and wash them clean.

[0050] (2) Making corn milk: Add 500g of corn and 500mL of water to the soy milk maker in a corn-to-water ratio of 1:1 and make corn milk using the "five grains" mode. After filtering, you will get about 550mL of corn milk.

[0051] (3) Filtration: First filter with 1 layer of medical gauze once, then filter with 4 layers of medical gauze once to obtain corn juice.

[0052] (4) Mix and homogenize: Add 70g of soybean flour and 70g of sugar to 460g of filtered corn juice, bring the volume to 1000mL and stir well. Homogenize at 1.2w speed for 5 minutes.

[0053] (5) Dispensing: Dispense the obtained corn milk into containers in 200mL portions.

[0054] (6) Sterilization: Place the packaged corn into a water bath at 80°C and sterilize for 15 minutes.

[0055] (7) Preparation of BFS1243 bacterial powder: Take 1 mL of BFS1243 bacterial suspension from a laboratory-preserved glycerol tube and add it to 4 mL of MRS liquid medium. Incubate at 37°C for 24 h to obtain a turbid bacterial suspension. After shaking, take an inoculation loop and streak the liquid onto a blank MRS plate. Incubate at 37°C for 12 h to obtain a single colony. Re-inoculate the single colony into 5 mL of MRS liquid medium and incubate at 37°C for 12 h. Take 1 mL of the obtained bacterial suspension and add it to 100 mL of MRS liquid medium. Incubate at 37°C for 12 h, then add it to a fresh 700 mL of MRS liquid medium and expand the culture at 37°C for 12 h. Centrifuge at 4°C for 14 min and discard the supernatant to obtain bacterial sludge. Take 1g of mycelium sludge and mix it thoroughly with 5mL of protectant (the protectant is a 5% maltodextrin solution). Pre-freeze at 4℃ for 1h, then transfer it to -20℃ for 1h, then transfer it to -80℃ for 12h, and finally freeze-dry it in a freeze dryer at -80℃ for 24h. The resulting mycelium powder is sealed and stored at -20℃.

[0056] (8) Activation and inoculation of the starter culture: The mixed starter culture stored at -20℃ was weighed in a clean bench at a volume of 0.002% of the yogurt volume to prepare the starter culture activation solution (the starter culture composition was: plant yogurt starter culture No. 1 (product of Guangzhou Miaodou Technology Co., Ltd.) and the above-prepared Lactobacillus plantarum BFS1243 starter culture at a mass ratio of 2:1), so that the total starter culture content in the final corn juice reached 0.02g / L and the number of viable bacteria per milliliter reached 2×10 6 CFU / mL.

[0057] (9) Fermentation: Place the corn juice at 30℃ and ferment for 10 hours.

[0058] (10) Post-fermentation and finished product storage: After fermentation, place the fermented corn milk at a constant temperature of 4℃ for 12-24 hours for post-fermentation. Store the fermented corn milk at 4℃ for 28 days to obtain black sweet corn yogurt.

[0059] Example 2: Effects of different starter cultures on the antioxidant activity, physicochemical properties, and sensory characteristics of black sweet corn yogurt. 1. Effects of different starter cultures on the antioxidant activity of yogurt Referring to the fermentation process in Example 1, black sweet corn yogurt was made using different starter cultures, and three different fermentation groups were set up: Group A: Inoculated with conventional fermentation agents: Plant yogurt starter No. 1 powder (CK); Group B: Inoculated with compound microbial agent: Plant yogurt starter No. 1 powder + Lactobacillus plantarum BFS1243 powder, with a mass ratio of 1:1; Group C: Inoculated with Lactobacillus plantarum BFS1243 bacterial powder and fermented separately.

[0060] Antioxidant activity determination at fermentation endpoint: The antioxidant activity and antioxidant substances were determined in the three experimental groups. Antioxidant activity was determined using a commercially available kit, while the method for determining antioxidant substances followed the specific methods outlined in the literature and national standards. (1) Determination of total flavonoids: Take 1 mL of yogurt sample into a 10 mL centrifuge tube, add 4 mL of 60% ethanol, sonicate at 50℃ for 30 min, centrifuge (4800 r / min, 10 min) to obtain the supernatant, and use the supernatant for the experiment. Referring to the method (Yang Qinru, Zhang Wenkai, Zheng Guodong. Optimization of ultrasonic extraction process of total flavonoids from Hibiscus root and study on in vitro antioxidant activity, 2025), take 1 mL of sample solution into a 5 mL centrifuge tube, add 300 uL of 5% NaNO2 solution, shake well and react for 6 min. Then add 300 uL of 10% Al(NO3)3 solution, shake well and react for another 6 min, then add 2 mL of 1 mol / L NaOH solution. After incubation at room temperature for 15 min, measure the absorbance at a wavelength of 510 nm. Plot a standard curve by fitting the concentration of rutin standard as the x-axis and the absorbance value as the y-axis through linear regression. The total flavonoid content of the sample was calculated using a regression equation, expressed as rutin equivalents (mgRE / 100g FW) per 100g fresh sample. The standard curve regression equation was obtained as y = 1.6595x + 0.0433 (R²). 2 =0.9984).

[0061] (2) Determination of total polyphenols: Take 1 mL of yogurt sample into a 10 mL centrifuge tube, add 4 mL of 75% ethanol, sonicate at 50℃ for 30 min, centrifuge (4800 r / min, 10 min) to obtain the supernatant, and use the supernatant for the experiment. The determination was carried out according to the national standard GB / T44349-2024, the determination of total polyphenols in bee pollen by the Folin-Ciocalteu reagent colorimetric method. After cooling to room temperature, centrifuge at 5000 r / min for 10 min. Take 0.2 mL of gallic acid standard solution of each concentration and the test solution into 5 mL centrifuge tubes, add 0.5 mL of Folin-Ciocalteu reagent to each, shake well and let stand for 3 min. Add 1.5 mL of 10.0% sodium carbonate solution to each, and let stand at room temperature in the dark for 20 min. Use 75% ethanol as a blank control and measure the absorbance at a wavelength of 765 nm. Plot a standard curve with the concentration of gallic acid standard as the abscissa and the absorbance value as the ordinate. The total polyphenol content of the sample was calculated using a regression equation, and expressed as gallic acid equivalent per 100g of fresh sample (mg / 100g). The regression equation for the standard curve was y = 6.7377x + 0.028 (R²). 2 = 0.9978).

[0062] (3) Determination of total anthocyanin: The method was slightly modified from that in (JL, Rw D, Re W. Determination of total monomeric anthocyanin pigment content of fruit juices, beverages, naturalcolorants, and wines by the ph differential method: collaborative study., 2006): 1.0 g of sample was placed in a 50 mL centrifuge tube, and 80% ethanol was added as the extraction solvent. The extraction was repeated multiple times under the conditions of a material-to-liquid ratio of 1:35, ultrasonic power of 105 W, extraction time of 30 min, and extraction temperature of 50 ℃. The extracts were then mixed and centrifuged at 4800 r / min for 10 min. The extract was then rotary evaporated at approximately 0.08 mPas-0.09 mPas at 50 ℃, and finally diluted to 10 mL to obtain a crude extract of corn anthocyanins. The extract was stored at 4 ℃ in the dark for later use. Add 4.5 mL of prepared HCl-KCl buffer (pH=1) and HCl-CH3COONa buffer (pH=4.5) to 10 mL centrifuge tubes, respectively, followed by 0.5 mL of crude anthocyanin extract. Measure the absorbance at 528 nm and 700 nm, respectively, and calculate the total anthocyanin content in the sample using the following formula. The anthocyanin content is expressed as cornflower equivalents.

[0063] Calculation formula: Total anthocyanin content (mg Cy-3-glu / g DW) = (A × MW × DF) / (ε × L × V); In the formula: A: (A528nm-A700nm)pH1.0-(A528nm-A700nm)pH4.5; MW: relative molecular mass of cyanidin, 449.2 g / mol; DF: Dilution factor 200; ε: molar absorptivity, 26900 L × mol -1 ×cm -1 ; L: The optical path of the 96 microplate is 0.5 cm; V: The volume of the sample measured, 0.2 mL; m: Dry weight of material, g.

[0064] The measurement results are as follows Figure 1 As shown in the figure, group C, which was fermented alone with BFS1243, showed a significant increase in DPPH scavenging rate of black sweet corn yogurt compared to group A after 18 hours of fermentation, from 84.64% to 85.94%. Group B, which used a compound microbial agent, showed a significant increase in antioxidant activity compared to the other two groups, with FRAP reducing power, ·OH oxygen free radical scavenging rate, and DPPH scavenging rate reaching 0.512, 36.87%, and 87.99%, respectively.

[0065] Furthermore, during fermentation, the content of antioxidant active substances, except for polyphenols, showed an increasing trend, while the content of the other two showed a decreasing trend. The changes in group C were significantly greater than those in group A. At the end of fermentation, group C had the lowest flavonoid and anthocyanin contents, at 20.67 mg / 100 mL and 69.56 mg / L respectively, while its total polyphenol content was the highest at 83.95 mg / 100 mL. Group B also performed exceptionally well, with a more significant decrease in flavonoid and anthocyanin contents than group A, decreasing to 23.98 mg / 100 mL and 76.04 mg / L respectively. The increase in total polyphenol content was significantly greater than that in group A and not significantly different from group C, reaching 81.39 mg / 100 mL. The results indicate that after fermentation using the compound group B, the total polyphenol content increased, while the contents of total flavonoids and total anthocyanins decreased after decomposition and transformation. Overall, the retention rate of anthocyanins was low, but the levels of small molecule phenolic acids and antioxidant activity were significantly improved.

[0066] 2. Statistical results of pH, viscosity, and total sensory score at the fermentation endpoint The pH and viscosity of the yogurt in the three fermentation groups were measured respectively, as follows: (1) pH: After stirring the yogurt sample evenly, measure the pH value with a pH meter and record the value after the reading stabilizes. Perform the operation in parallel for each sample 3 times and take the average value.

[0067] (2) Viscosity: The yogurt sample was placed for 30 min to return to room temperature. 30 mL was measured and poured into a 50 mL centrifuge tube. The viscosity was measured and recorded at different rotor speeds (using rotor No. 3, with speeds set to 12, 30, and 60 r / min respectively). The viscosity coefficient C was calculated according to the following formula and used to characterize the viscosity value of the sample.

[0068] Calculation formula:

[0069]

[0070] In the formula: V: shear rate during viscometer measurement, s -1 ; w: Angular velocity of the rotor during the viscometer measurement, in rad / s; r: rotational speed, rpm; Rb: Rotor radius, mm; Rc: Inner diameter of the container, mm; A: The constant that converts angular velocity into shear rate; η: viscosity, mPa·s; B: Viscosity coefficient, mPa·s.

[0071] (3) Sensory evaluation: Refer to (Yang Liping, Zheng Shulin, Long Xiaoshan, et al. Development and quality analysis of corn yogurt products [J]. Journal of Jinggangshan University (Natural Science Edition), 2022, 43(03):47-53.) with slight adjustments: 7-9 food professionals were randomly invited to conduct sensory evaluation, evaluating the appearance, texture, smell and taste of different samples. Each person tasted one sample at a time, and rinsed their mouth with purified water after tasting to ensure the accuracy of the evaluation. The maximum and minimum values ​​were removed from the scores, and the results were expressed as mean ± standard deviation. The sensory evaluation criteria for black sweet corn yogurt are shown in Table 1.

[0072] Table 1 Sensory rating table for black sweet corn yogurt

[0073] (4) Measurement results: pH measurement results of different fermentation groups are as follows Figure 2As shown, group C, fermented with BFS1243 alone in a black sweet corn plant emulsion substrate, exhibited better acid production performance, reaching a significantly lower pH of 4.15 at the 18-hour fermentation endpoint. In contrast, group B, fermented with the combined strain, had a pH of 4.37, while group A only reached 4.76. This indicates that both single and combined fermentation using the BFS1243 strain demonstrate superior acid production capabilities, enabling faster attainment of the pH standard (4.6-4.8) for commercially available yogurt. Specifically, fermentation using plant-based yogurt starter culture No. 1 alone required 18 hours, fermentation using BFS1243 powder alone required 12 hours, and combined fermentation only required 10 hours.

[0074] Viscosity measurement results of different fermentation groups Figure 3 As shown, group B, which underwent compound fermentation, exhibited the highest viscosity at 5771.35 mPa·s, followed by group A at 4442.5 mPa·s, while group C only reached 3235.59 mPa·s. This demonstrates that while single-strain fermentation produces superior acid, excessively low pH levels cause soybean protein to deviate from its isoelectric point, resulting in a thinner consistency and lacking the richness expected of yogurt, thus affecting its sensory appeal. Further sensory evaluation results for each group, such as... Figure 4 As shown, the sensory scores of group C are significantly lower than those of groups A and B, indicating that fermentation of BFS1243 strain alone leads to a single acidity and poor texture, resulting in a decline in the sensory quality of yogurt. Compared with the existing starter culture, the yogurt fermented by group A has a better texture and richer flavor, but the fermentation time is longer. Combining BFS1243 strain with the existing plant-based yogurt starter culture No. 1 can rapidly reduce the pH to the fermentation endpoint, shorten the fermentation time (effectively shortening it by 8 hours) while retaining the rich flavor and excellent texture of the plant-based yogurt starter culture No. 1.

[0075] Example 3: Effect of compound microbial inoculants on changes in anthocyanins and phenolic acids in black sweet corn yogurt Based on the research results of Example 2, further comparative fermentation experiments were set up: Group A: inoculated only with plant-based yogurt starter culture No. 1 (CK); Group B: inoculated with plant-based yogurt starter culture No. 1 + Lactobacillus plantarum BFS1243 culture (mass ratio of 1:1, i.e., compound group). The initial latex of black sweet corn that did not undergo fermentation was used as the initial control before fermentation. The changes in anthocyanins and phenolic acids were monitored during the fermentation process. High performance liquid chromatography (HPLC) was used to perform targeted quantitative determination of anthocyanins and phenolic acids in the black sweet corn yogurt sample liquid from 0-18 h after inoculation. The determination method is as follows: (1) Determination of anthocyanins: An Agilent Poroshell (C18 column, 4.6 x 100 mm, 2.7 μm) chromatographic column was used at a column temperature of 30 °C. Mobile phase: Phase A: 2% formic acid aqueous solution (w / v); Phase B: acetonitrile, flow rate 1 mL / min, gradient set as follows: 0-55 min, Phase B ratio 5-83%; 55-60 min, Phase B ratio 83-5%. Injection volume 10 μL, DAD detector was used, acquisition wavelength 520 nm. A standard curve was established using standards for quantitative analysis, and the content was expressed in mg / L. Changes in the content and peak area of ​​each anthocyanin component during 18 h of fermentation were recorded.

[0076] (2) Determination of phenolic acids: An Agilent Poroshell (C18 column, 4.6 x 100 mm, 2.7 μm) column was used at a column temperature of 30 °C. Mobile phase: A: methanol: water: formic acid (10:88:2 v / v); B: methanol: water: formic acid (90:8:2 v / v), flow rate 1 mL / min, gradient set as follows: 0-20 min, B phase ratio 0-15%; 20-30 min, B phase ratio 15-50%; 30-35 min, B phase ratio 50-100%; 35-42 min, B phase ratio 100-0%. Injection volume 10 μL, DAD detector was used, acquisition wavelength 320 nm. A standard curve was established using standards for quantitative analysis, and the content was expressed in mg / L. The changes in the content of each component of phenolic acids during 18 h of fermentation were recorded.

[0077] The results of anthocyanin content determination are as follows: Figure 5 As shown in Table 2, in the initial latex of unfermented black sweet corn, anthocyanins mainly exist as macromolecular glycosides of cyanidin, pelargonidin, and paeoniflorin, and their acylated derivatives. After 18 hours of fermentation, anthocyanins in group B underwent significant degradation and transformation, with the total anthocyanin content decreasing to 76.04 mg / L (significantly lower than 98.70 mg / L in group A). ​​Furthermore, the structural composition changed significantly. As shown in Table 2, the proportion of acylated anthocyanins in group B decreased significantly from 53.75% to 49.71%, and the proportion of pelargonidin derivatives decreased significantly from 16.23% to 10.79%. These data indicate that the addition of BFS1243 greatly promoted the decomposition of macromolecularly bound anthocyanins.

[0078] Table 2. Proportion of various anthocyanins after fermentation and at the initial stage in different experimental groups.

[0079] Note: * indicates a significant difference from the initial group, * indicates a significant difference, and ** indicates a highly significant difference.

[0080] The results of phenolic acid content and total phenol changes showed that, along with the targeted degradation of macro-molecular anthocyanins, the chromatographic peaks representing small-molecule phenolic acids in the liquid chromatogram of group B showed a significant increase, as shown in Table 3. This indicates that the contents of p-hydroxybenzoic acid, p-coumaric acid, trans-cinnamic acid, and other phenolic acids in group B showed a significant upward trend. This suggests that the compound fermentation using BFS1243 can further convert macro-molecular anthocyanins into highly active small-molecule phenolic acids, solving the problems of easy degradation and low bioavailability of anthocyanins in black sweet corn.

[0081] Table 3. Content of phenolic acids in each experimental group at different fermentation times.

[0082] Simultaneously, whole-genome sequencing (WGS) analysis of *Lactobacillus plantarum* BFS1243 revealed a large number of GH1 family β-glucosidase genes, as shown in Table 4. Further correlation analysis of changes in the content of various substances during fermentation was also conducted. Figure 6 As shown, it can be concluded that: under the action of highly active β-glucosidase specifically secreted by BFS1243, the glycosidic bonds of macromolecular anthocyanins (such as cyanidin-3-glucosidase) are precisely cleaved, and the sugar moiety is removed to form anthocyanin aglycones; subsequently, under the action of esterases of BFS1243, they are further cleaved and metabolized, and targeted to be converted into free phenolic acids with small molecular weights such as p-hydroxybenzoic acid, p-coumaric acid and trans-cinnamic acid.

[0083] Table 4. Details of BFS1243 Carbohydrate Active Enzyme Analysis

[0084] Example 4: Addition and Screening of Stabilizers Because the acid production rate increases after adding BFS1243 bacterial powder, and the gelation speed of plant-based proteins (mainly soy protein) in yogurt increases after the fermentation time is shortened, and the corn starch in the system ages during storage at 4℃, the gel stability of the compounded plant protein is poor. In order to further solve the stability problem of black sweet corn plant-based yogurt after fermentation, five common food-grade hydrocolloids were added as stabilizers in the homogenization of step (4) of Example 1: soluble soybean polysaccharide, xanthan gum, guar gum, low-ester pectin and carrageenan. At the same time, two addition gradients of 0.1% (low dose) and 0.3% (high dose) were set for different stabilizers. According to the preparation method of Example 1, black sweet corn yogurt with different stabilizers was prepared (the fermentation agent used was plant yogurt starter powder No. 1 + Lactobacillus plantarum BFS1243 bacterial powder, with a mass ratio of 1:1). At the same time, fermented yogurt without stabilizers was used as a control. The yogurts prepared separately were stored under refrigeration conditions, and their water holding capacity and apparent viscosity were measured. The viscosity was measured using the same method as in Example 2. The water-holding capacity was measured as follows: 5g of black sweet corn yogurt was placed in a 10mL centrifuge tube and centrifuged at 4000r / min for 20min. The supernatant was removed, and the mass of the residue and the centrifuge tube was measured.

[0085] Calculation formula: Water holding capacity = m1 / (m2-m0); In the formula, m0 is the mass (g) of a 10mL centrifuge tube, m1 is the mass (g) of yogurt before centrifugation, and m2 is the mass (g) of soy yogurt and centrifuge tube after centrifugation.

[0086] The measurement results are as follows Figure 7 As shown, the experimental groups that added low-ester pectin, carrageenan, or guar gum to the fermentation feed showed no significant improvement in water-holding capacity, and at high doses, the yogurt became excessively viscous and hard, leading to severe whey separation. Xanthan gum and soluble soybean polysaccharides, on the other hand, demonstrated better process adaptability in improving water-holding capacity (reducing whey separation) and maintaining suitable viscosity. Specifically, using 0.1% and 0.3% xanthan gum maintained water-holding capacity at 45.93% and 55.98% at 28 days, respectively, while using 0.1% and 0.3% soybean polysaccharides maintained water-holding capacity at 56.04% and 55.98% at 28 days. In contrast, the group without any stabilizers only maintained a water-holding capacity of 44.18% at 28 days. Furthermore, the addition of stabilizers can improve viscosity. 0.3% xanthan gum maintained the viscosity at 4362.80 mPa·s at 28 days, 0.1% soybean polysaccharide maintained the viscosity at 2888.76 mPa·s at 28 days, while the viscosity of the group without any stabilizers was only 2601.29 mPa·s at 28 days.

[0087] The combined results of water-holding capacity and viscosity show that using soluble soybean polysaccharide as a stabilizer can better maintain the water-holding capacity of yogurt. Due to its unique three-dimensional molecular structure, soybean polysaccharide can not only effectively stabilize the soybean protein and corn system through steric hindrance, but also does not cause excessive increase in the viscosity of the system, thus improving the stability of plant protein gel.

[0088] Experiment Example 5: Determination of the Storage Stability of Black Sweet Corn Yogurt Based on the study in Example 4, soluble soybean polysaccharides were selected as a stabilizer and added at different amounts: 0.01%, 0.05%, 0.10%, 0.15%, and 0.20%. Storage stability tests were conducted, with the combined fermentation group without stabilizer serving as a control. The water-holding capacity, viscosity, and other physicochemical properties of the yogurt were measured at storage periods of 0, 5, 10, 15, and 20 days, and sensory evaluation was performed for a comprehensive assessment. Specific measurement methods and standards were referenced in Examples 2 and 4.

[0089] The results of the water-holding capacity and viscosity measurements are as follows: Figure 8 and Figure 9 As shown, with the addition of soluble soybean polysaccharides from 0.01% to 0.20%, the water-holding capacity increased from 52.75% to 54.63% and the viscosity from 2282 mPa·s to 2892.41 mPa·s after 20 days. However, when the addition amount was ≥0.10%, there was no significant difference in the increase of water-holding capacity and viscosity; at a 0.10% addition amount, the water-holding capacity and viscosity remained at 56.04% and 2888.76 mPa·s after 20 days, respectively. Meanwhile, the sensory quality evaluation results are as follows... Figure 10 As shown, the sensory quality of fermented yogurt decreased less over 20 days, with the 0.10% addition level performing best overall and scoring highest. At this addition level, the black sweet corn plant-based yogurt exhibited excellent water retention throughout its shelf life, with no significant whey separation or pigment stratification. Its texture was also smooth and delicate, maximizing the preservation of the aroma and flavor of black sweet corn and the release of active substances such as small-molecule phenolic acids, significantly extending the product's stable shelf life. Conversely, in the yogurt group without stabilizers, whey separation and stratification occurred around 14 days, severely affecting appearance. The compounded plant protein gel exhibited poor stability and a shorter shelf life.

[0090] In summary, this invention provides a preparation process for plant-based yogurt rich in phenolic acids from black sweet corn. This method uses black sweet corn juice and soybean flour as a matrix, and employs a compound fermentation agent containing *Lactobacillus plantarum* strain BFS1243 for compound fermentation. The resulting black sweet corn yogurt exhibits increased total polyphenol content and high levels of antioxidant active substances. The addition of *Lactobacillus plantarum* BFS1243 converts the large-molecule anthocyanins in black sweet corn into highly active small-molecule phenolic acids, primarily p-hydroxybenzoic acid and p-coumaric acid, thus solving the problems of easy degradation and low bioavailability of anthocyanins in black sweet corn. The resulting yogurt is a bright and stable pink color with a delicate texture, effectively masking the beany smell and possessing a unique corn aroma. Its antioxidant activity (DPPH, FRAP, ·OH scavenging rate) and total phenolic content are significantly superior to traditional fermented products. Furthermore, this invention replaces coconut powder with black sweet corn juice in the original plant-based (soybean-coconut powder) yogurt, which can better improve the flavor quality, giving it a unique corn aroma and pink appearance, and also has good antioxidant activity. At the same time, by adding specific stabilizers, it can further solve the problem of poor stability of compound plant protein gel, and extend the shelf life of black sweet corn plant-based yogurt.

[0091] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. A method for preparing black sweet corn plant-based yogurt rich in phenolic acids, characterized in that, Includes the following steps: S1: Mix black sweet corn juice, soybean flour, and white sugar with water, homogenize to obtain a plant-based emulsion, sterilize and set aside; the mass ratio of black sweet corn juice: soybean flour: white sugar: water is (30~70):(5~10):(5~10):(10~60). S2: A compound fermentation agent is added to the sterilized plant-based emulsion, and fermentation is carried out under constant temperature conditions to obtain fermented curd; the compound fermentation agent contains plant-based yogurt starter No. 1 powder and Lactobacillus plantarum (… Lactobacillus plantarum BFS1243 strain, with a mass ratio of (1~5):1; S3: After fermentation, the curd is refrigerated and then matured to obtain black sweet corn plant-based yogurt rich in phenolic acids.

2. The preparation method according to claim 1, characterized in that, The total viable count of plant-based yogurt starter powder No. 1 in S2 is not less than 1.0 × 10⁻⁶. 9 CFU / g, the total viable count of strain BFS1243 is not less than 1.0 × 10⁻⁶. 9 CFU / g.

3. The preparation method according to claim 2, characterized in that, A stabilizer is also added to the raw materials of S1, and the mass ratio of the black sweet corn juice to the stabilizer is (400~500):(1~3).

4. The preparation method according to claim 3, characterized in that, The stabilizer is selected from carrageenan, soluble soybean polysaccharide, low-ester pectin, guar gum, or xanthan gum.

5. The preparation method according to claim 4, characterized in that, The stabilizer is a soluble soybean polysaccharide.

6. The preparation method according to claim 2, characterized in that, The sterilization conditions in S1 are: water bath sterilization at 60~90℃ for 10~20 minutes.

7. The preparation method according to claim 2, characterized in that, The fermentation temperature in S1 is 25~35℃, and the fermentation time is 8~18 hours.

8. The preparation method according to claim 2, characterized in that, The conditions for cold-curing in S3 are: curing at 0~5℃ for 12~24 hours.

9. A black sweet corn plant-based yogurt rich in phenolic acids prepared by the method according to any one of claims 1 to 8.

10. Lactobacillus plantarum ( Lactobacillus plantarum The application of strain BFS1243 in the preparation of black sweet corn plant-based yogurt as described in claim 9, or in promoting the conversion of macromolecular anthocyanins into small molecule phenolic acids in black sweet corn, is characterized by, The BFS1243 strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on November 5, 2022, with accession number GDMCC No:62947.

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