Preparation method of multi-grain coarse cereal pancake based on complex microbial inoculants

Abstract

The invention discloses a preparation method of a multi-grain coarse grain pancake based on a complex microbial inoculant. According to the method, the fermentation process of the pancake is improved synergistically through complex microbial inoculant screening and experimental design optimization. Through synergistic interaction among the three dominant strains, a substrate can be decomposed more sufficiently, the fermentation efficiency is improved, and the production of flavor substances is improved. Meanwhile, the alpha-amylase activity and the sensory score of the pancake serve as double optimization indexes, key parameters are accurately regulated and controlled, and it is guaranteed that the fermentation efficiency and the final product quality are synchronously improved. A synergistic system of microbial agent screening and experimental design optimization is formed, the fermentation period is remarkably shortened, the sensory score is improved, a standardized production technology of the coarse cereal pancake is provided, and finally the high-quality pancake which is rich in nutrition, unique in flavor and excellent in quality is stably and efficiently produced. A scientific and reliable technical scheme is provided for standardized, industrialized and high-quality production of the multi-grain coarse grain pancakes, and the method has a good application prospect.

Description

Technical Field

[0001] This invention relates to the field of food processing technology, and in particular to a method for preparing multigrain pancakes based on a compound microbial agent. Background Technology

[0002] Traditional methods use corn, millet, soybeans, and other grains as raw materials, grinding them into a fine paste with water, and then spreading it at high temperature for a short time. The resulting pancakes can be as thin as a cicada's wing, resilient yet not hard, or thick and soft, combining portability and long shelf life. Rich in carbohydrates, dietary fiber, and plant protein, these pancakes provide efficient energy, enhance satiety, promote intestinal peristalsis, and maintain stable blood sugar levels, making them a low-fat, healthy staple food with significant development potential.

[0003] However, the pancake industry faces several technological bottlenecks in its modernization process. Traditional production processes generally rely on commercial yeast fermentation, lacking specific screening of particular strains. This results in long fermentation cycles, low fermentation efficiency, monotonous flavor, coarse texture, and low sensory scores, failing to meet consumer demands for high-quality food. Furthermore, the limitations of single-strain fermentation in enzyme composition, metabolites, and functional characteristics lead to insufficient nutrient conversion and difficulty in enhancing functional properties. In addition, existing pancake fermentation processes are largely based on experience-based adjustments, lacking standardized production techniques and precise process control, resulting in unstable product quality and failing to meet the demands of standardization, industrialization, and high-quality production.

[0004] Therefore, there is an urgent need in this field for a new method that can deeply integrate targeted and efficient microbial agent screening with scientific experimental design methods to overcome the blindness and inefficiency of existing technologies, and ultimately stably and efficiently produce high-quality pancakes that are rich in nutrients, have a unique flavor, and are of excellent quality. Summary of the Invention

[0005] The purpose of this invention is to provide a method for preparing multigrain pancakes based on compound microbial agents, which solves the problems of single microbial strains, long fermentation cycle, low fermentation efficiency, insufficient nutrient conversion, single flavor, low sensory score and unstable process parameters in the traditional multigrain pancake fermentation process.

[0006] To solve the above-mentioned technical problems, the present invention adopts the following technical solution: a method for preparing multigrain pancakes based on compound microbial agents, comprising the following steps:

[0007] 1) Mix the activated Lactobacillus fermentation liquid, Saccharomyces cerevisiae liquid and Pediococcus pentosaceus liquid, then centrifuge to collect the cells, and resuspend the obtained cells in an equal volume (liquid) of physiological saline to obtain a compound bacterial agent;

[0008] 2) Grind the multigrain mixture with water into a fine paste and adjust the pH to 6.1-6.4 to obtain the pancake batter. Then, inoculate the pancake batter with the compound microbial agent obtained in step 1) to ensure that the total number of live bacteria is not less than 10. 8 ~10 10 The mixture is fermented at CFU / mL at 29-33℃ for 3-5 hours, then spread out to obtain multigrain pancakes with high α-amylase activity and sensory scores. The multigrain pancakes include rice, corn, millet, and soybeans. This utilizes a synergistic system of "microbial agent compounding—microbial purification—fermentation regulation." Fermentation with a compound microbial agent using three dominant strains—Lactobacillus, Saccharomyces cerevisiae, and Pediococcus pentosaceus—effectively avoids the limitations of single-strain fermentation in traditional processes by leveraging their synergistic interactions in acid production, gas production, and flavor compound generation. pH control and fermentation temperature control create a suitable microenvironment for the synergistic effect of the microbial agents, ultimately producing pancakes with high α-amylase activity and sensory scores in the shortest possible fermentation time, achieving a systematic improvement in the nutritional quality and sensory flavor of the pancakes.

[0009] Preferably, the activation of the *Lactobacillus fermentum* broth, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus* involves the following steps: freeze-dried *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus* powders are inoculated into liquid culture medium for cultivation. After separation and purification, single colonies are picked and cultured in liquid culture medium for one generation. Then, the first-generation culture is inoculated into new liquid culture medium for a second generation until the above-mentioned strains reach their maximum growth rate, thus obtaining activated *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus* broths. The second-generation culture time for *Lactobacillus fermentum* is 4.5 hours, and the second-generation culture time for *Saccharomyces cerevisiae* and *Pediococcus pentosaceus* is 6.5 hours. This solves the problem of poor microbial activity and batch stability in traditional processes, ensuring high efficiency at the start of subsequent fermentation and reliable results.

[0010] Preferably, the liquid culture medium for the brewing yeast is YPD, the culture temperature is 30℃, and the culture time is 16-24 hours; the liquid culture medium for the *Lactobacillus fermentum* and *Pediococcus pentosaceus* is MRS, the culture temperature is 37℃, and the culture time is 16-24 hours. This ensures that the strains reach their peak activity before being inoculated into the pancake batter, which is a prerequisite for rapid and efficient fermentation. It also solves the problem of lack of standardized and professional operation in traditional microbial agent activation processes, ensuring high standards and consistency in microbial agent activity.

[0011] Preferably, the conditions for centrifuging to collect bacterial cells are: centrifugation at 8000~10000 r / min for 6~15 min.

[0012] Preferably, the mass ratio of rice, corn, millet, and soybeans is 12:5:2:1. This ratio, determined through experimental optimization, fully reflects the nutritional value of multigrain cereals while ensuring the rheological properties and substrate balance of the pancake batter, making it optimal for the synergistic fermentation of the compound microbial agent. A reasonable substrate ratio is fundamental to ensuring efficient microbial metabolism and the flavor of the final product.

[0013] Preferably, the volume ratio of the Lactobacillus fermentation culture, Saccharomyces cerevisiae culture, and Pediococcus pentosaceus culture is 1:1:1. This ratio results in strong synergistic effects between the microorganisms, promoting α-amylase synthesis, thorough starch decomposition, and balanced flavor compound production, leading to excellent sensory qualities in the pancake. Simultaneously, it effectively avoids competition or excessive inhibition between dominant microorganisms, which could disrupt metabolic balance, reduce activity, and consequently affect the pancake's texture and aroma.

[0014] Preferably, before grinding into a paste, the multi-grain raw materials are pretreated as follows: rice is steamed; corn, millet, and soybeans are soaked for 4-5 hours; then the cooked materials are mixed evenly with the soaked raw materials and left to stand for 1-2 hours; the mass ratio of grains to water in the pancake batter is 1:2-5. In this way, steaming (gelatinizing) the rice and soaking (softening) the other grains effectively break down the starch structure, increase its hydration, and make subsequent grinding finer. It also improves the usability of starch and other substrates for enzymatic decomposition by microbial agents. Mixing and standing further homogenizes the materials, ensuring the uniformity of the pancake batter and solving the problems of difficult grinding, low substrate utilization, and coarse pancake texture caused by coarse raw material processing in traditional processes.

[0015] Preferably, the fermentation conditions are: a fermentation temperature of 33℃ and a fermentation time of 5 hours. This fermentation temperature is optimal for the synergistic growth and metabolism of the three microbial strains, achieving peak α-amylase activity and sensory evaluation scores. This fermentation time minimizes the fermentation cycle while maximizing enzyme activity and sensory evaluation scores, thus achieving a perfect balance between fermentation efficiency and product quality.

[0016] Preferably, the inoculation amount is 2-3% of the pancake batter volume. Thus, the inoculation amount is a key factor affecting the fermentation start-up speed and competition among microorganisms. An inoculation amount within the 2-3% range ensures a sufficient initial number of microorganisms in the system, stimulating the synthesis and secretion of α-amylase, while avoiding excessive resource competition caused by excessively high cell density.

[0017] Preferably, the multigrain pancake has an α-amylase activity of 98-124 U / g and a sensory score of 85-92. Thus, α-amylase activity represents the degree of starch degradation and nutrient release, while the sensory score represents the degree of improvement in flavor and texture. This demonstrates that the prepared pancake meets the requirements for high quality.

[0018] Another object of the present invention is to provide a multigrain pancake obtained by the above method. By solidifying all the preferred microbial agents and process parameters into the final product, the pancake is ensured to have unparalleled α-amylase activity, excellent microstructure, rich flavor compounds, and the highest sensory score compared to ordinary pancakes.

[0019] Compared with the prior art, the present invention has the following beneficial effects:

[0020] 1. This invention provides a method for synergistically improving the fermentation process of pancakes through a compound microbial agent and optimized experimental design. By using a compound of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, it effectively avoids the limitations of single-strain microorganisms in terms of enzyme composition, metabolites, and functional characteristics. Furthermore, *Lactobacillus fermentum* rapidly produces acid, creating an acidic environment that inhibits the growth of other microorganisms, ensuring fermentation safety. Simultaneously, the acidic environment is conducive to the subsequent metabolism and enzyme activity of *Pediococcus pentosaceus* and *Saccharomyces cerevisiae*; *Saccharomyces cerevisiae* efficiently utilizes the substrate, producing various flavor compounds and enhancing sensory flavor. Its produced enzymes also synergistically decompose starch; *Pediococcus pentosaceus* has a strong gas-producing capacity, and the generated gas can be evenly distributed in the batter, forming a stable bubble structure. This not only results in high fermentation activity of the pancake batter but also endows the final pancake with excellent characteristics of being soft, delicious, and having a loose and delicate texture. The synergistic interaction of these three components enables more thorough substrate decomposition, improves fermentation efficiency, and enhances the generation of flavor compounds, collectively achieving a systematic improvement in both the fermentation efficiency and final quality of the pancakes. Furthermore, through single-factor and orthogonal experiments, fermentation time, temperature, inoculum size, inoculum ratio, and alkali neutralization pH were systematically optimized, achieving precise control of key parameters in the fermentation process. Using the α-amylase activity and sensory evaluation scores of the pancakes as dual optimization indicators, the simultaneous improvement of fermentation efficiency and final product quality was ensured. This invention achieves a systematic improvement in the nutritional quality, functional characteristics, and sensory flavor of pancakes.

[0021] 2. The preparation method of this invention is simple and easy to operate, with low production costs and high fermentation efficiency, greatly reducing the time and energy costs of industrial production. The fermentation cycle is significantly shortened from the traditional 12 hours to the optimal 5 hours, greatly improving fermentation efficiency. The sensory score of the prepared multigrain pancakes increases from the traditional 70% to over 85%, achieving a significant improvement of over 21.43%. This invention systematically solves the efficiency and quality problems in the fermentation of multigrain pancakes.

[0022] 3. This invention provides a standardized production technology for multigrain pancakes, ensuring stable product quality. It ultimately enables the stable and efficient production of high-quality pancakes that are nutritious, have a unique flavor, and are of excellent quality. This invention provides a scientific and reliable technical solution for the standardized, industrialized, and high-quality production of multigrain pancakes and has good application prospects. Attached Figure Description

[0023] Figure 1 This is a growth curve diagram for different strains.

[0024] Figure 2 The time for secondary subculturing of different strains and the number of viable cells at the point of maximum activity.

[0025] Figure 3 This is a graph showing the changes in pH and total acidity during the fermentation of pancake batter by different bacterial strains.

[0026] Figure 4 This study illustrates the changes in the number of viable bacteria during fermentation of pancake batter by different bacterial strains over varying fermentation time.

[0027] Figure 5 The fermentation activity of different strains of fermented pancake batter was studied.

[0028] Figure 6 Microscopic structures of pancake batter fermented by different strains.

[0029] Figure 7 The effect of fermentation time of compound microbial agents on α-amylase activity in pancake batter and sensory score of pancakes.

[0030] Figure 8 The effect of fermentation temperature of compound microbial agents on α-amylase activity in pancake batter and sensory scores of pancakes.

[0031] Figure 9 The effect of the inoculum amount of the compound microbial agent on the α-amylase activity of pancake batter and the sensory score of pancakes.

[0032] Figure 10 The effect of the ratio of microbial strains in a compound microbial agent on the α-amylase activity of pancake batter and the sensory score of pancakes.

[0033] Figure 11 The effect of alkali neutralization pH on α-amylase activity and sensory scores of pancake batter. Detailed Implementation

[0034] The present invention will be further described in detail below with reference to the embodiments. Unless otherwise specified, all reagents used in the embodiments are commercially available.

[0035] Example 1: Screening of fermentation strains

[0036] 1) Determination of bacterial growth curve and viable cell count

[0037] Inoculation: 1% freeze-dried bacterial powders of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, *Pediococcus pentosus*, and *Leuconostoc mesenteroides* were cultured in liquid culture medium in a clean bench to obtain bacterial suspensions. *Saccharomyces cerevisiae* was cultured in YPD liquid medium at 2000 r / min and 30℃ in a shaker for 16-24 h. *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Bifidobacterium lactis*, *Pediococcus pentosus*, and *Leuconostoc mesenteroides* were cultured in MRS broth medium in an incubator at 37℃ for 16-24 h.

[0038] Isolation and purification: The streak plate method was used for isolation and purification. Six bacterial cultures were streaked onto solid culture media using a disposable inoculation loop in three zones to gradually disperse the strains into individual colonies. Saccharomyces cerevisiae was cultured on yeast extract peptone glucose agar and incubated upside down at 30°C for 24-36 h. Other strains were cultured on MRS agar and incubated upside down at 37°C for 24-36 h.

[0039] First subculture: Single colonies from solid culture media were picked and cultured in corresponding liquid culture media. Saccharomyces cerevisiae was cultured in YPD liquid medium in a shaker at 2000 r / min and 30℃ for 16-24 h. Lactobacillus plantarum, Lactobacillus fermentum, Bifidobacterium lactis, Pediococcus pentosus, and Leuconostoc mesenteroides were cultured in MRS broth medium in an incubator at 37℃.

[0040] Second subculturing: The bacterial cultures from the first subculture were inoculated at a 1% inoculum into the corresponding optimal liquid culture medium for second-generation culture. The growth curves of the six bacterial strains from 0 to 24 h were determined using a turbidimetric method, with uninoculated liquid culture medium as a blank control. The OD values ​​of the culture media were measured at 600 nm using a microplate reader from 0 to 24 h. Three replicates were set up for each group, and growth curves for each strain were plotted. The results are shown below. Figure 1 As shown.

[0041] Depend on Figure 1 It can be seen that during the cultivation process, the bacterial density of different strains initially increased and then stabilized over time, which is consistent with the general law of microbial growth. From 0 to 3 hours, the microbial growth is slow; the bacteria need to adapt to the environment and initiate metabolism, resulting in minimal reproduction and a gradual increase in bacterial density. From 3 to 12 hours, the microbial growth enters the logarithmic phase, with rapid bacterial proliferation. *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Bifidobacterium lactis* show similar but steeper growth slopes, while *Saccharomyces cerevisiae*, *Pediococcus pentosaceus*, and *Leuconostoc mesenteroides* show similar but gentler growth slopes. After 12 hours, all strains enter the stationary phase, and due to limited resources in the culture system, the bacterial density tends to be similar.

[0042] Furthermore, the stage with the largest slope in the logarithmic growth curve of the strain indicates vigorous metabolism, high activity, excellent stress resistance, stable genetic material, and rapid adaptation to new environments, representing the peak activity of the strain. The viable cell count at this peak activity was then determined using the dilution plating method. Specifically, yeast was diluted with physiological saline, and lactic acid bacteria were diluted with peptone-salt solution. Four dilutions were set up with three replicates, and the cultures were incubated for 1-2 days. When the colony count was between 30 and 300, the viable cell count was calculated. The results are as follows: Figure 2 As shown.

[0043] Depend on Figure 2 It was found that the peak activity times varied among different bacterial strains. The peak activity times for *Lactobacillus plantarum*, *Lactobacillus fermentum*, and *Bifidobacterium lactis* was 4.5 hours, while those for *Saccharomyces cerevisiae*, *Pediococcus pentosaceus*, and *Leuconostoc mesenteroides* was 6.5 hours. Therefore, these times can be selected as the culture time for the second subculture of each strain. Furthermore, the viable cell counts of *Saccharomyces cerevisiae*, *Pediococcus pentosaceus*, and *Leuconostoc mesenteroides* were significantly higher than those of the other strains when their activity was at its peak.

[0044] 2) Determination of pH and total acidity during the fermentation of pancake batter

[0045] Weigh rice, cornmeal, millet, and soybeans in a ratio of 12:5:2:1 and rinse three times. Add 1.5 times the weight of water to the rice and cook in a rice cooker for 30 minutes. Soak the cornmeal, millet, and soybeans in water for 4 hours. After the cooked mixture cools, mix the raw and cooked mixtures with twice the weight of water, stir well, and let stand for 1 hour. Grind the settled mixture into a fine paste using a colloid mill to obtain the pancake batter. Centrifuge the second-generation culture of each bacterial strain and resuspend it in an equal volume of physiological saline. Then, inoculate each strain into the pancake batter at an inoculum volume of 3% of its volume, controlling the fermentation temperature at 33℃.

[0046] The pH values ​​of the pancake batter were measured using a benchtop pH meter at 0, 2, 4, 6, and 8 hours of fermentation. Simultaneously, based on the principle of acid-base neutralization, the total acidity was determined using potentiometric titration with a pH meter. 10 g of fermented pancake batter was mixed with 90 ml of distilled water, and the pH of the sample was titrated with 0.1 mol / L NaOH until it reached 8.5. The volume (mL) of NaOH standard titration solution consumed was recorded as the total titration acidity (TTA). The results are as follows: Figure 3 As shown.

[0047] Depend on Figure 3It can be seen that as fermentation progresses, the pH value of each fermentation system continuously decreases, while the total acidity continuously increases. At 4 hours, the pH value of most pancake batters has dropped below 5, indicating that the strains have good fermentation effects. Within 0-8 hours, the pH value of *Lactobacillus fermentatus* decreases significantly more than other strains, indicating its more prominent metabolic intensity and better utilization of the substrate, thus improving pancake quality. From 0-4 hours, the pH value of *Lactobacillus fermentatus* decreases rapidly, quickly initiating acid-producing metabolism and regulating the environmental pH to the inhibition range for harmful bacteria. From 6-8 hours, the pH value of *Lactobacillus fermentatus* is at its lowest and continues to decrease, demonstrating its tolerance to acidic environments. Therefore, when using pH and total acidity as indicators, *Lactobacillus fermentatus* should be selected as the dominant strain for fermenting pancake batter.

[0048] 3) Determination of viable bacteria count during pancake batter fermentation

[0049] As above, activated second-generation bacterial cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, *Pediococcus pentosaceus*, and *Leuconostoc mesenteroides* were inoculated into the pancake batter for fermentation. The fermentation temperature was controlled at 33℃, and the inoculum size was 3%. The viable cell counts of the pancake batter at 0, 2, 4, 6, and 8 hours of fermentation were determined using the dilution plating method. The results are as follows: Figure 4 As shown.

[0050] Depend on Figure 4 It can be seen that the viable cell count of each strain in the fermented pancake batter generally showed an increasing trend with the extension of time. From 0 to 4 hours, the increase in viable cell count of each strain was relatively slow, indicating that they were in the adaptation and initial proliferation stage. From 4 to 8 hours, they entered the rapid proliferation period, and the viable cell count increased significantly. Among them, Saccharomyces cerevisiae showed the greatest increase, exhibiting explosive growth, indicating that it has high utilization efficiency of the pancake batter substrate, can more fully decompose the pancake batter components, improve fermentation efficiency, and generate flavor substances. Therefore, when using the viable cell count of the pancake batter as an indicator, Saccharomyces cerevisiae should be selected as the dominant strain for fermenting the pancake batter.

[0051] 4) Determination of fermentation activity of pancake batter

[0052] As above, activated second-generation bacterial cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, *Pediococcus pentosus*, and *Leuconostoc mesenteroides* were inoculated into the pancake batter. 60 ml of the pancake batter with the added cultures was placed in a 250 mL graduated cylinder and fermented at 33°C. The volume change of the pancake batter before and after 8 hours of fermentation was recorded, and the fermentation activity of the pancake batter was calculated. The results are as follows: Figure 5 As shown. The formula for calculating fermentation activity is as follows:

[0053] (1)

[0054] Depend on Figure 5It is evident that *Lactobacillus plantarum* exhibits extremely low fermentation activity, indicating a weak ability to produce gas and promote volume change during fermentation. The fermentation activities of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, and *Leuconostoc mesenteroides* are similar, all around 0.33. *Pediococcus pentosacchari*, however, demonstrates a fermentation activity reaching 1, doubling the system volume during fermentation. Based on fermentation activity, *Pediococcus pentosacchari* exhibits significantly higher gas production capacity than other strains, more efficiently driving substrate decomposition and system expansion during fermentation, thus contributing to the soft and delicious characteristics of the pancakes. Therefore, when using the fermentation activity of the pancake batter as an indicator, *Pediococcus pentosacchari* should be selected as the dominant strain.

[0055] 5) Determination of the microstructure of pancake batter

[0056] As above, activated second-generation bacterial cultures of *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, *Pediococcus pentosaceus*, and *Leuconostoc mesenteroides* were inoculated into the pancake batter for fermentation. The fermentation temperature was controlled at 33℃, the fermentation time was 5 hours, and the inoculation amount of the bacterial culture was 3%. After fermentation, the microstructure of the pancake batter was observed using an electron microscope and photographed. The results are as follows: Figure 6 As shown.

[0057] Depend on Figure 6 It was found that the pancake batter fermented by *Lactobacillus plantarum*, *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, *Bifidobacterium lactis*, and *Leuconostoc mesenteroides* had relatively few bubbles and a dispersed distribution, while the batter fermented by *Pediococcus pentosacchari* had abundant and evenly distributed bubbles. This indicates that *Pediococcus pentosacchari* can efficiently produce gas and form a superior bubble structure during fermentation. These bubbles make the pancakes more porous and delicate in texture during preparation, and are also beneficial for the retention and release of flavor compounds. Therefore, when using the microstructure of the pancake batter as an indicator, *Pediococcus pentosacchari* should be selected as the dominant strain.

[0058] In summary, this invention uses five core indicators—batter pH, total acidity, viable cell count, microstructure, and fermentation activity—to accurately assess the potential of microbial strains in pancake fermentation, effectively avoiding the blind spots of traditional screening methods. The selected compound microbial agents—Lactobacillus fermentum, Saccharomyces cerevisiae, and Pediococcus pentosaceus—work synergistically when used in combination, enabling more thorough substrate decomposition, improved fermentation efficiency, and enhanced flavor compound generation, collectively achieving a systematic improvement in both pancake fermentation efficiency and final quality.

[0059] Example 2: Optimization of Fermentation Process Conditions for Compound Microbial Agents

[0060] Determination of α-amylase activity in pancake batter: Weigh 2.0 g of batter, add α-amylase extraction buffer to 30 ml, and extract at room temperature for 20 min, shaking every few minutes. Then centrifuge at 8000 r / min for 10 min and collect the supernatant, which is the enzyme solution. Take 1.0 ml of the enzyme extract and heat it in a 70℃ constant temperature water bath for 15 minutes to inactivate β-amylase. Add 1.0 ml of pH 5.6 citrate buffer to each tube, and add 4.0 ml of NaOH (0.4 N) to the control tube to inactivate the enzyme activity. Incubate each tube in a 40℃ constant temperature water bath for 15 min, add 2.0 mL of starch solution preheated at 40℃, shake well, and immediately place in a 40℃ water bath for 5 min. Remove and add 4.0 ml of NaOH (0.4 N) to the test tube to terminate the enzyme activity. Add 2.0 ml of the above enzymatic reaction solution to 2.0 ml of DNS and boil in a water bath for 5 min. Dilute with distilled water to 25 ml. Measure the absorbance at 540 nm using a microplate reader. Establish a standard curve using maltose, expressed as A = 0.2411x + 0.0047 (where A is absorbance, x (mg / mL) is the reducing sugar concentration in the reaction system, and R² = 0.9868).

[0061] The formula for calculating enzyme activity is as follows:

[0062] (2)

[0063] Where: C is the reducing sugar concentration (mg / mL) obtained from the standard curve; V is the total volume of the reaction solution (mL); n is the sample dilution factor; m is the mass of the solid sample weighed (g); t is the reaction time (min); and M is the molar mass of maltose (g / mol).

[0064] Sensory evaluation criteria for pancakes: The samples to be evaluated were placed in a 25°C constant temperature laboratory. A judging panel of 10 students majoring in food-related fields was selected. Judges were required to abstain from alcohol, smoking, and spicy or irritating foods for 12 hours prior to the test. Judges evaluated the samples individually without discussion. After evaluating one sample, there was a 5-minute pause before moving on to the next. The sensory evaluation criteria are shown in Table 1.

[0065] Table 1 Sensory evaluation criteria for multigrain pancakes fermented with mixed cultures

[0066]

[0067] 1. Single-factor experimental design

[0068] 1) Effects of fermentation time on α-amylase activity in pancake batter and sensory scores of pancakes

[0069] After second-generation culture of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, the resulting bacterial solutions were mixed at a volume ratio of 1:1:1, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected and resuspended in an equal volume of physiological saline to obtain a compound bacterial agent. This compound bacterial agent was then inoculated into pancake batter (prepared using the same method as in Example 1, hereinafter the same) for fermentation. The fermentation temperature was controlled at 33℃, the pH value was neutralized with alkali at 6.1, and the inoculum size was 3.0%. The effects of fermentation times of 1 h, 2 h, 3 h, 4 h, and 5 h on pancake quality were investigated. The results are as follows: Figure 7 As shown.

[0070] Depend on Figure 7 It was observed that both α-amylase activity and sensory scores showed a continuous upward trend with prolonged fermentation time, reaching their maximum at 5 hours. From 1 to 3 hours, the microorganisms were in an adaptation phase, resulting in low metabolic activity and minimal amylase secretion. From 3 to 5 hours, the microorganisms began to proliferate rapidly, and the synergistic effect between strains was enhanced, accelerating enzyme activity. This facilitated the decomposition of starch and other substrates in the pancake batter, improving the pancake texture and releasing flavor compounds, thus simultaneously boosting the sensory score. From 4 to 5 hours, as enzyme activity reached a certain limit, the decomposition of starch and other substrates became more complete, and the changes gradually slowed down.

[0071] 2) Effects of fermentation temperature on α-amylase activity in pancake batter and sensory scores of pancakes

[0072] After second-generation culture of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, the resulting bacterial solutions were mixed at a volume ratio of 1:1:1, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The cells were then resuspended in an equal volume of physiological saline to obtain a compound bacterial agent. This compound agent was then inoculated into pancake batter for fermentation. The fermentation time was controlled at 3 h, the pH was neutralized to 6.1, and the inoculum size was 3.0%. The effects of fermentation temperatures of 21℃, 25℃, 29℃, 33℃, and 37℃ on pancake quality were investigated. The results are as follows: Figure 8 As shown.

[0073] Depend on Figure 8 It can be seen that as the fermentation temperature increases, both α-amylase activity and sensory score show a trend of first increasing and then decreasing, with the best effect observed at a fermentation temperature of 33℃. Between 25 and 33℃, the temperature is suitable for the growth and metabolism of the microorganisms, promoting a rapid increase in amylase activity and simultaneously improving the quality of the pancakes; these two factors show a synergistic positive change. Between 33 and 37℃, α-amylase activity decreases rapidly with increasing temperature, indicating that excessively high temperatures inhibit enzyme activity and simultaneously worsen the sensory quality of the pancakes; these two factors show a synergistic negative change.

[0074] 3) Effects of inoculum size on α-amylase activity in pancake batter and sensory scores of pancakes

[0075] After second-generation culture of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, the resulting bacterial solutions were mixed at a volume ratio of 1:1:1, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The cells were then resuspended in an equal volume of physiological saline to obtain a composite bacterial solution. This composite bacterial solution was then inoculated into pancake batter for fermentation. The fermentation temperature was controlled at 33℃, the fermentation time at 3 h, and the pH value at alkali neutralization at 6.1. The effects of inoculum amounts of 1.5%, 2.0%, 2.5%, 3.0%, and 3.5% (v / w) on pancake quality were investigated. The results are as follows: Figure 9 As shown.

[0076] Depend on Figure 9 It was observed that as the inoculum size increased, both amylase activity and the sensory score of the pancakes showed a trend of first increasing and then decreasing, with the best effect observed at an inoculum size of 3.0%. At an inoculum size of 1.5%, amylase activity was affected by the number of bacteria, resulting in low amylase secretion and low activity. At inoculum sizes of 2.0%–3.0%, the large initial number of bacteria in the system stimulated more α-amylase synthesis and secretion, leading to an increase in enzyme activity, but the rate of increase slowed down. This may be due to the high bacterial density, intensified resource competition, and a decrease in the metabolic rate of some bacteria. At inoculum sizes of 3.0%–3.5%, enzyme activity decreased, possibly due to the high bacterial density, intensified resource competition, and a decrease in the metabolic rate of some bacteria.

[0077] 4) Effects of inoculation ratio on α-amylase activity in pancake batter and sensory score of pancakes

[0078] After second-generation culture of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, the resulting bacterial solutions were mixed at a specific volume ratio. The mixture was centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected and resuspended in an equal volume of physiological saline to obtain a compound bacterial agent. This compound agent was then inoculated into pancake batter for fermentation. The fermentation temperature was controlled at 33℃, the fermentation time at 3 h, the pH value at alkali neutralization at 6.1, and the inoculum size at 3.0%. The effects of volume ratios of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus* of 1:1:1, 2:1:1, 1:2:1, 1:1:2, and 1:3:1 on pancake quality were investigated. The results are as follows: Figure 10 As shown.

[0079] Depend on Figure 10It can be seen that with changes in the inoculation ratio, α-amylase activity and sensory scores generally showed a synergistic change, both exhibiting a trend of first decreasing, then increasing, and then decreasing again. At an inoculation volume ratio of 1:1:1, both performed relatively optimally, because at this ratio, the synergistic interaction between the microorganisms was strong, which was conducive to α-amylase synthesis, sufficient starch decomposition, balanced flavor compound generation, and excellent sensory qualities in the pancake. At inoculation volume ratios of 2:1:1, 1:2:1, and 1:1:2, competition or inhibition among dominant microorganisms intensified, metabolic balance was disrupted, amylase synthesis was inhibited, and activity decreased, severely affecting the pancake's texture and aroma, and lowering the sensory score. At an inoculation volume ratio of 1:3:1, the community structure was excessively disordered, the negative effects of interspecific interactions became prominent, and enzyme activity and sensory scores further declined.

[0080] 5) Effects of alkali neutralization pH on α-amylase activity in pancake batter and sensory scores of pancakes.

[0081] After second-generation culture of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, the resulting bacterial solutions were mixed at a volume ratio of 1:1:1, centrifuged at 8000 rpm for 10 min, and the bacterial cells were collected. The cells were then resuspended in an equal volume of physiological saline to obtain a composite bacterial solution. This composite bacterial solution was then inoculated into pancake batter for fermentation. The fermentation temperature was controlled at 37℃, the fermentation time at 3 h, and the inoculum size at 3.0%. The effects of alkaline neutralization pH values ​​of 5.8, 6.1, 6.4, 6.7, and 7.0 on pancake quality were investigated. The results are as follows: Figure 11 As shown.

[0082] Depend on Figure 11 It is observed that as the pH value increases after alkali neutralization, both α-amylase activity and sensory scores first increase and then decrease. At a pH value around 6.1, a balance can be struck between high amylase activity and good sensory quality of the pancakes, meeting the requirements for pancake batter preparation. pH 6.1 is close to the optimal environment for α-amylase, with a stable enzyme protein conformation, easy binding of the active site to the substrate, and high catalytic efficiency. This pH value is also suitable for the growth and metabolism of microorganisms in the pancake batter, which is conducive to the production of amylase, improves pancake quality, and promotes higher sensory scores. At a pH value of 7.0, the enzyme conformation changes, the active site is damaged, and the catalytic ability decreases. At the same time, it affects the growth and metabolism of microorganisms, resulting in a deterioration in sensory indicators such as pancake flavor and texture. Therefore, α-amylase activity and sensory scores decrease simultaneously.

[0083] 2. Orthogonal experimental design

[0084] Orthogonal optimization experiments were conducted based on the results of single-factor experiments. The factors and levels of the orthogonal experiments are shown in Table 2.

[0085] Table 2. Factors and Levels in Orthogonal Experiments

[0086]

[0087] Based on the results of the single-factor experiments, an L18(37) orthogonal experiment was conducted on five factors: fermentation time (A), fermentation temperature (B), inoculum quantity (C), inoculum ratio (D), and alkali neutralization pH (E), with α-amylase activity of fermented pancake batter and sensory score of pancakes as indicators. The results are shown in Table 3.

[0088] Table 3 Orthogonal Experimental Design and Results

[0089]

[0090] Table 3 shows that, based on the range (R), the order of influence of each factor on the overall score is C (inoculum size) > A (fermentation time) > B (fermentation temperature) > D (inoculum ratio) > E (neutralization pH value), with the inoculum size having the most significant impact. Based on the mean (k), the optimal combination is A3B3C3D3E3, which corresponds to a fermentation time of 5 h, a fermentation temperature of 33℃, an inoculum size of 3%, an inoculum ratio of 1:1:1, and a neutralization pH value of 6.1.

[0091] Table 4 Results of Analysis of Variance

[0092]

[0093] The results of the ANOVA in Table 4 show that the inoculum size has a highly significant impact on the fermentation effect, while fermentation time, fermentation temperature, inoculum ratio, and neutralization pH value have significant effects on the fermentation effect. The model mean square is much larger than the error mean square, and the error sum of squares accounts for a low proportion of the total sum of squares, indicating high experimental reliability, a high model fit to the data, and a reasonable experimental design.

[0094] In summary, this invention utilizes single-factor and orthogonal experiments to systematically optimize fermentation time, temperature, inoculum size, inoculum ratio, and alkali neutralization pH. Furthermore, it employs α-amylase activity and sensory evaluation scores of the pancake batter as dual optimization indicators, significantly improving the fermentation efficiency and sensory quality of the pancake batter. This provides a scientifically reliable technical solution for the standardized, industrialized, and high-quality production of multigrain pancakes.

[0095] Example 3: Preparation method of multigrain pancakes based on compound microbial agents

[0096] 1) Weigh 1% freeze-dried Lactobacillus fermentum, Saccharomyces cerevisiae, and Pediococcus pentosaceus into liquid culture medium in a clean bench and culture them. Saccharomyces cerevisiae is cultured in YPD liquid medium at 2000 r / min and 30℃ for 16-24 h. Lactobacillus fermentum and Pediococcus pentosaceus are cultured in MRS broth medium at 37℃ for 16-24 h. The bacterial solutions of Lactobacillus fermentum, Saccharomyces cerevisiae, and Pediococcus pentosaceus are obtained respectively.

[0097] 2) Use a disposable inoculation loop to dip the bacterial solution from step 1) into a solid culture medium and streak it in three zones to gradually disperse the strains into individual colonies. Saccharomyces cerevisiae was cultured on yeast extract peptone glucose agar and incubated upside down in a 30°C incubator for 24-36 h. Lactobacillus fermentum and Pediococcus pentosaceus were cultured on MRS agar and incubated upside down in a 37°C incubator for 24-36 h.

[0098] 3) Select single colonies from the solid culture medium and culture them in the corresponding liquid culture medium for 16-24 hours to obtain first-generation cultures of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*. Then, inoculate the first-generation cultures of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus* into new liquid culture medium at an inoculation rate of 1%, with *Lactobacillus fermentum* cultured for 4.5 hours and *Saccharomyces cerevisiae* and *Pediococcus pentosaceus* cultured for 6.5 hours to obtain second-generation cultures of *Lactobacillus fermentum*, *Saccharomyces cerevisiae*, and *Pediococcus pentosaceus*, i.e., activated bacterial solutions.

[0099] 4) Mix the activated bacterial solutions of Lactobacillus fermentum, Saccharomyces cerevisiae and Pediococcus pentosus in a volume ratio of 1:1:1, then centrifuge at 8000 r / min for 10 min, discard the supernatant, wash the bacterial sludge with physiological saline by repeated centrifugation, and finally resuspend in an equal volume of physiological saline to obtain the compound bacterial agent.

[0100] 5) Weigh the rice, cornmeal, millet, and soybeans in a weight ratio of 12:5:2:1 and rinse them three times. Then, add 1.5 times the weight of water to the rice and cook it in a rice cooker for 30 minutes. Soak the cornmeal, millet, and soybeans in water for 4 hours. After the cooked ingredients have cooled, mix them with the raw ingredients and twice the weight of water, stir well, and let it stand for 1 hour. Finally, grind the settled ingredients into a fine paste using a colloid mill, and neutralize the pH to 6.1 with alkali to obtain the pancake batter.

[0101] 6) Inoculate the compound microbial agent obtained in step 4) into the pancake batter at 3% of the pancake batter volume and ferment at 33℃ for 5 h.

[0102] 7) Preheat the pancake maker, spray cooking oil with an oil bottle, spread it evenly with an oil rubber, pour the fermented pancake batter vertically into the center of the pan, and immediately spread it clockwise with a scraper using your wrist as the axis to ensure that the thickness and area of ​​the pancake are approximately the same, thus obtaining the pancake.

[0103] The results showed that the α-amylase activity of the multigrain pancake in this embodiment was 124.103 U / g, the sensory score was 92.119, and the comprehensive score was 108.111, ensuring the simultaneous improvement of fermentation efficiency and final product quality.

[0104] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing multigrain pancakes based on a compound microbial agent, characterized in that, Includes the following steps: 1) The activated Lactobacillus fermentation liquid, Saccharomyces cerevisiae liquid and Pediococcus pentosus liquid were mixed separately, then the cells were collected by centrifugation, and the obtained cells were resuspended in an equal volume of physiological saline to obtain a compound bacterial agent; 2) Grind the multigrain mixture with water into a fine paste and adjust the pH to 6.1-6.4 to obtain the pancake batter. Then, inoculate the pancake batter with the compound microbial agent obtained in step 1) to ensure that the total number of live bacteria is not less than 10. 8 ~10 10 The mixture is fermented at 29-33℃ for 3-5 hours and then spread out to obtain a multigrain pancake with high α-amylase activity and sensory score; the multigrain includes rice, corn, millet and soybean.

2. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The activation of the Lactobacillus fermentation culture, Saccharomyces cerevisiae culture, and Pediococcus pentosaceus culture is carried out using the following steps: Freeze-dried Lactobacillus fermentum, Saccharomyces cerevisiae, and Pediococcus pentosaceus were inoculated into liquid culture medium for culture. After isolation and purification, single colonies were picked and cultured in liquid culture medium for the first generation. Then, the first generation culture was inoculated into new liquid culture medium for the second generation culture until the above strains reached their maximum growth rate, thus obtaining activated Lactobacillus fermentum, Saccharomyces cerevisiae, and Pediococcus pentosaceus cultures. The second generation culture time for Lactobacillus fermentum was 4.5 hours, and the second generation culture time for Saccharomyces cerevisiae and Pediococcus pentosaceus was 6.5 hours.

3. The method for preparing multigrain pancakes based on compound microbial agents according to claim 2, characterized in that, The liquid culture medium for the brewing yeast is YPD, the culture temperature is 30℃, and the culture time is 16~24h; the liquid culture medium for the Lactobacillus fermentum and Pediococcus pentosus is MRS, the culture temperature is 37℃, and the culture time is 16~24h.

4. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The conditions for collecting bacterial cells by centrifugation are: centrifugation at 8000~10000 r / min for 6~15 min.

5. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The mass ratio of rice, corn, millet and soybean is 12:5:2:1; the volume ratio of the mixed Lactobacillus fermentation liquid, Saccharomyces cerevisiae liquid and Pediococcus pentosaceus liquid is 1:1:

1.

6. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, Before grinding into a paste, the multigrain mixture is pretreated as follows: rice is steamed to obtain cooked material; corn, millet and soybeans are soaked for 4-5 hours to obtain raw material; then the cooked material and the soaked raw material are mixed evenly and left to stand for 1-2 hours; the mass ratio of multigrain mixture to water in the pancake batter is 1:2-5.

7. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The fermentation conditions are: fermentation temperature of 33℃ and fermentation time of 5h.

8. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The inoculation amount of the compound microbial agent is 2-3% of the volume of the pancake batter.

9. The method for preparing multigrain pancakes based on compound microbial agents according to claim 1, characterized in that, The α-amylase activity of the multigrain pancake was 98~124U / g, and the sensory score was 85~92.

10. Multigrain pancakes obtained by the method according to any one of claims 1 to 9.

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