A compound microbial inoculant and application thereof in preparation of chicken liver flavoring

Abstract

The present application relates to the technical field of microbial inoculant, and particularly relates to a compound microbial inoculant and application of the compound microbial inoculant in preparation of chicken liver flavor seasoning, wherein the compound microbial inoculant is prepared by mixing Lactobacillus plantarum YR07 Lactiplantibacillus plantarum ) and Saccharomyces cerevisiae HQSAJ-001-24 Saccharomyces cerevisiae ) in a volume ratio of 1:1-3, wherein the Lactobacillus plantarum YR07 7 and the Saccharomyces cerevisiae HQSAJ-001-24 are respectively activated and cultured to a viable bacterial count of greater than or equal to 1x10 7 CFU / mL. Through the synergistic effect of the compound microbial inoculant fermentation and Maillard reaction, the inherent fishy smell of chicken liver is completely covered and transformed, bitter peptides are deeply degraded, umami and sweet amino acids are enriched, a compound system containing multiple key flavor substances is constructed, and finally a high-quality natural seasoning with rich flavor levels, fresh and mellow taste and significant flavor enhancement function is prepared.

Description

Technical Field

[0001] This invention relates to the field of microbial inoculants, specifically to a compound inoculant and its application in the preparation of chicken liver flavored seasoning. Background Technology

[0002] Chicken liver is a major byproduct of poultry processing, rich in protein, amino acids, and various minerals, possessing high nutritional value and development potential. However, chicken liver has a strong fishy smell, a coarse texture, and a high cholesterol content, limiting its direct consumption or application in high-value-added foods. Currently, chicken liver is mostly processed into animal feed, resulting in low utilization and resource waste. Therefore, developing technologies for the high-value utilization of chicken liver has significant economic and social implications.

[0003] In the field of seasonings, the preparation of flavor enhancers from animal protein hydrolysates via the Maillard reaction has become an important technical method. Chicken liver, after enzymatic hydrolysis, yields hydrolysates rich in small peptides and free amino acids, which can serve as excellent substrates for the Maillard reaction. However, chicken liver protein hydrolysates obtained through traditional enzymatic hydrolysis processes still suffer from noticeable bitterness and a limited flavor profile, directly impacting their applicability as seasoning ingredients.

[0004] For example, traditional chicken liver powder is made from fresh chicken liver through physical processing steps such as washing, steaming or baking, dehydration, and pulverization. Its core technology lies in drying and pulverizing the chicken liver through heat processing and physical crushing, aiming to retain its nutritional components and extend its shelf life. However, traditional chicken liver powder products have a strong fishy smell and a limited flavor. Because it relies solely on heat processing, it not only fails to effectively remove the inherent fishy odor of chicken liver, but also exhibits a weak Maillard reaction, resulting in a lack of rich meaty and nutty aromas and other appealing flavors, leading to poor sensory quality and limited application.

[0005] For example, enzymatically hydrolyzed chicken liver powder is made by adding an enzymatic hydrolysis step before dehydration and pulverization, based on traditional chicken liver powder processing. Typically, proteases (such as trypsin) are used to catalyze the hydrolysis of cooked chicken liver pulp, aiming to degrade large protein molecules into smaller peptides and amino acids to improve solubility and enhance flavor. However, enzymatically hydrolyzed chicken liver powder has a noticeable bitter taste and insufficient flavor modification: while proteolysis can increase flavor compounds, it exposes hydrophobic amino acids, resulting in a significant and persistent bitterness. At the same time, the simple enzymatic hydrolysis process has limited optimization of flavor precursors, failing to effectively enrich umami and sweet amino acids, leading to insufficient umami and poor flavor harmony in the product. Summary of the Invention

[0006] To address the problems of traditional chicken liver powder having a strong fishy smell and a monotonous flavor, enzymatically hydrolyzed chicken liver powder having a noticeable bitter taste and insufficient umami flavor, as well as the shallow flavor profile and limited functionality of existing products, this invention provides a compound microbial agent and its application in the preparation of chicken liver flavored seasonings.

[0007] This invention utilizes the synergistic effect of compound microbial fermentation and Maillard reaction to completely mask and transform the inherent fishy smell of chicken liver, deeply degrade bitter peptides and enrich umami and sweet amino acids, thereby constructing a complex system containing multiple key flavor substances, and finally preparing a high-quality chicken liver-derived natural seasoning with rich flavor layers, delicious and mellow taste and significant flavor enhancement function.

[0008] Specifically, the present invention provides the following technical solution:

[0009] A compound microbial agent, composed of Lactobacillus plantarum YR07 ( Lactiplantibacillus plantarumum ) and brewer's yeast HQSAJ-001-24 ( Saccharomyces cerevisiae Activate and culture separately until the viable bacterial count is ≥ 1×10⁻⁶. 7 CFU / mL, mixed in a volume ratio of 1:1-3;

[0010] The Lactobacillus plantarum YR07 ( Lactiplantibacillus plantarum It is deposited at the China Center for Type Culture Collection on August 18, 2022, with the biological accession number CCTCC NO:M20221303;

[0011] The brewing yeast HQSAJ-001-24 ( Saccharomyces cerevisiae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2025, with the biological accession number CGMCC No. 35983.

[0012] This compound microbial agent is the core component for achieving efficient degradation of bitter peptides, enrichment of sweet amino acids, and generation of key flavor precursors.

[0013] The compound microbial agent of the present invention can be used to prepare chicken liver flavored seasoning, specifically including the following steps:

[0014] (1) Preparation of chicken liver protein hydrolysate (CLPH): Fresh or frozen chicken liver is used as raw material. After washing, removing blood vessels and bile ducts, and homogenizing, it is heated at 85-90℃ for 15-20 minutes to inactivate enzymes. Then, it is defatted with 50-60% isopropanol at a material-to-liquid ratio of 1:4–1:6 (w / v). After centrifugation, the precipitate is collected to obtain chicken liver protein. The chicken liver protein is reconstituted in water, the pH is adjusted to 7.5-8.5, and trypsin is added at 1% of the substrate weight. The mixture is hydrolyzed at 40℃ for 2-4 hours, and then the enzyme is inactivated at 85-90℃ for 10-15 minutes to obtain chicken liver protein hydrolysate.

[0015] (2) Fermentation of compound microbial agent: Inoculate the chicken liver protein hydrolysate obtained in step (1) with 2% total inoculum of compound microbial agent, add 5% glucose at the same time, adjust the initial pH to 6.0-7.0, ferment at 30℃ for 36-48 hours, and then terminate the fermentation by heat treatment at 85-90℃ for 10-15 minutes to obtain fermented chicken liver protein hydrolysate.

[0016] (3) Maillard reaction flavor enhancement: Xylose (1.5-2.5% of the dry basis weight of the hydrolysate), L-cysteine ​​hydrochloride (0.5-1.5%), and thiamine hydrochloride (0.03-0.07%) are added sequentially to the fermented modified chicken liver protein hydrolysate obtained in step (2). After stirring evenly, the pH is adjusted to 6.5-7.5, and the mixture is placed in a reaction vessel and reacted at 115-125℃ and a stirring speed of 150-250 rpm for 60-120 minutes. The reaction is terminated immediately by cooling in an ice bath after the reaction is completed. This Maillard reaction substrate (hereinafter referred to as MR substrate) is the decisive factor for the efficient generation of key flavor compounds such as methylthiopropionaldehyde and pyrazines, which are specific meat and nut flavor compounds.

[0017] (4) Product post-processing and preparation: The Maillard reaction product obtained in step (3) is centrifuged at 8000 rpm for 10-15 minutes, the supernatant is collected, and then spray dried at an inlet air temperature of 170-190℃ and an outlet air temperature of 80-90℃ to obtain the final chicken liver flavor seasoning powder product.

[0018] The final chicken liver flavored seasoning contains the following characteristic components and contents: total protein content ≥60% (dry basis), total free amino acid content ≥35 mg / g, of which umami amino acids (aspartic acid, glutamic acid) account for ≥25%, and sweet amino acids (glycine, alanine, serine) account for ≥30%; among the key flavor substances, the total amount of pyrazine compounds ≥80 μg / kg, the total amount of furan compounds ≥100 μg / kg, and sulfur-containing compounds (calculated as methylthiopropional) ≥120 μg / kg. This product has a significant meaty aroma, nutty aroma, and rich umami flavor, and can be used in compound seasonings, instant seasoning packets, meat products, and soup bases for flavor enhancement.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] (1) Existing technologies do not involve the microbial compound fermentation process, resulting in a lack of flavor substances such as organic acids and esters produced by fermentation in the products, leading to low flavor complexity. At the same time, they also fail to significantly improve the flavor intensity, richness, and natural flavoring function of the products through the synergistic effect of fermentation and Maillard reaction. However, the compound microbial agent of the present invention can use chicken liver as raw material, and through compound fermentation, it can further decompose proteins, reduce the content of bitter peptides, and produce flavor precursors such as organic acids and esters, providing a richer matrix for the subsequent Maillard reaction. In turn, it can synergistically prepare flavor seasonings with the Maillard reaction, so as to realize the high-value utilization of chicken liver by-products and improve the natural flavor quality of seasonings.

[0021] (2) The chicken liver flavoring prepared by this invention effectively eliminates the fishy smell of chicken liver and constructs a rich and harmonious complex flavor. Due to the use of a combined fermentation process of Lactobacillus plantarum and Saccharomyces cerevisiae, microbial metabolism effectively transforms the fishy smell precursors in chicken liver; subsequently, through Maillard reaction using xylose, L-cysteine ​​and thiamine as substrates, key flavor compounds such as methylthiopropionaldehyde (meaty aroma), pyrazines (nutty aroma, roasted aroma) and 2,3-butanedione (creamy aroma) are directionally generated. The test results show that the content of the above key flavor substances in the final product is significantly higher than that of products made by traditional processes, thereby fundamentally masking the off-flavor of the raw materials and forming a characteristic flavor with rich layers and prominent meaty aroma.

[0022] (3) This invention significantly improves the flavor quality of the product, solving the problems of prominent bitterness and insufficient umami. In the compound fermentation step, the synergistic effect of microorganisms deeply hydrolyzes proteins, especially effectively degrading hydrophobic bitter peptides produced by enzymatic hydrolysis. At the same time, the fermentation process optimizes the composition profile of free amino acids, increasing the proportion of umami amino acids (aspartic acid and glutamic acid) in the total free amino acids to more than 25%, the proportion of sweet amino acids (glycine, alanine, etc.) to more than 30%, while the content of bitter amino acids (such as leucine and isoleucine) is significantly reduced (it was determined that the content of bitter amino acids after fermentation can be reduced by up to 51.9%). This optimization of flavor precursors, combined with the further enhancement of flavor by the Maillard reaction, makes the final product exhibit excellent flavor characteristics of rich umami, natural sweetness, and virtually no unpleasant bitterness.

[0023] (4) This invention achieves the targeted preparation and efficient conversion of flavor precursors through a sequential process of "enzymatic hydrolysis-compound fermentation-Maillard reaction". Compared with the process of single enzymatic hydrolysis or single fermentation followed by direct Maillard reaction, the seasoning prepared by this invention has a total volatile flavor content increased by more than 50%, and the types and quantities of key aroma active substances (OAV>1) are greater. Electronic tongue analysis shows that its umami and sweetness response values ​​are significantly higher than those of the control sample. This makes the product not only rich and long-lasting in flavor, but also has a significant flavor-enhancing function. When added to food as a natural seasoning, it can effectively improve the overall flavor profile of the product.

[0024] Biological Preservation

[0025] Lactobacillus plantarum YR07, classified and named Lactiplantibacillus plantarum It is deposited at the China Center for Type Culture Collection (CCTCC), located at No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, on August 18, 2022, with accession number CCTCC NO: M20221303.

[0026] Saccharomyces cerevisiae HQSAJ-001-24, classified and named Saccharomyces cerevisiae It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. The deposit date is September 16, 2025, and the accession number is CGMCC No. 35983. Attached Figure Description

[0027] Figure 1 The pH values ​​are for CLP, CLPH, FCLPH, and their MRPs. CLP represents chicken liver protein, CLPH represents chicken liver protein hydrolysate, FCLPH represents fermented chicken liver protein hydrolysate, CLPM represents chicken liver protein Maillard reaction products, CLPPHM represents chicken liver protein hydrolysate Maillard reaction products, and FCLPHM represents fermented chicken liver protein hydrolysate Maillard reaction products.

[0028] Figure 2 The browning intensity and absorbance of intermediate products of CLP, CLPH, FCLPH and their MRPs are given.

[0029] Figure 3 The particle size of CLP, CLPH, FCLPH and their MRPs.

[0030] Figure 4 The free amino content of CLP, CLPH, FCLPH and their MRPs.

[0031] Figure 5Radar image of electronic nose data for CLP, CLPH, FCLPH and their MRPs.

[0032] Figure 6 Radar graph of electronic tongue data for CLP, CLPH, FCLPH and their MRPs.

[0033] Figure 7 The free amino acid content of CLP, CLPH, FCLPH and their MRPs.

[0034] Figure 8 Correlation analysis of sensory properties (E-tongue and E-nose) of CLP, CLPH, FCLPH and their MRPs with 35 volatile flavor compounds ( p ≤0.05). Detailed Implementation

[0035] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0036] I. Materials and Reagents

[0037] Frozen chicken livers were purchased from Qingdao Tianrun Food Co., Ltd. (Shandong, China), transported to the laboratory via cold chain, and stored at -20℃ for later use. *Lactobacillus plantarum* YR07 ( Lactiplantibacillus plantarum ) and brewer's yeast HQSAJ-001-24 ( Saccharomyces cerevisiae The strains were isolated from naturally fermented sausage and sake, respectively, both of which exhibit excellent flavor-producing characteristics. The strains were identified by sequencing of the 16S rRNA gene (bacteria) and the ITS region (yeast).

[0038] II. Preparation of Chicken Liver Flavored Seasoning

[0039] 1. Preparation of Chicken Liver Protein Hydrolysate (CLPH)

[0040] After thawing frozen chicken livers, surface blood clots were washed away, and larger blood vessels and bile ducts were removed. The processed chicken livers were homogenized to obtain a chicken liver homogenate. The homogenate was heated at 85 °C for 20 min to inactivate endogenous enzymes. 50% isopropanol was added for defatting at a solid-liquid ratio of 1:5 (w / v), and the mixture was vortexed and allowed to stand for 20 min. The mixture was then centrifuged at 4 °C and 4000 rpm for 15 min, and the supernatant was removed to obtain chicken liver protein (CLP). 100 g of the residue was mixed with distilled water at a ratio of 1:3 (w / v) and named the CLP group. The pH of the mixture was then adjusted to 8.0 with 0.5 M NaOH solution. Protein hydrolysis was performed using Trypsin for 3 h. The hydrolysate was heated at 85 °C for 20 min to inactivate the enzymes and named the CLPH group. The CLP group underwent the same treatment steps as the CLPH group, except that Trypsin was not added. Samples from both the CLP and CLPH groups were temporarily stored at -20 °C for subsequent fermentation.

[0041] 2. Compound fermentation treatment of CLPH

[0042] Lactobacillus plantarum YR07, preserved in 25% (v / v) glycerol solution Lactiplantibacillus plantarum ) and brewer's yeast HQSAJ-001-24 ( Saccharomyces cerevisiae The bacteria were inoculated into sterile MRS broth and YPD medium, respectively. *Lactobacillus plantarum* was cultured at 37 °C for 24 h, and *Saccharomyces cerevisiae* was cultured at 30 °C for 24 h. The activation process was repeated twice (two generations) until the viable cell concentration reached approximately 1 × 10⁻⁶. 7 CFU / mL. The culture medium was centrifuged at 4 ℃ and 8000 rpm for 10 min to collect the bacterial cells, and the supernatant was discarded. The bacterial pellet was washed three times with sterile physiological saline, resuspended in sterile physiological saline of the same volume as the initial volume, and stored at 4 ℃ for later use (within 24 h).

[0043] The frozen CLPH was thawed, and glucose was added to a final concentration of 6% (w / v). The pH was adjusted to 6.5 with 0.5 M NaOH solution. The mixture was sterilized at 85°C for 20 minutes. The activated *Lactobacillus plantarum* suspension and *Saccharomyces cerevisiae* suspension were mixed at a volume ratio of 1:2 and inoculated into the sterilized CLPH substrate at a total inoculation rate of 2% (v / v). Fermentation was carried out in a constant temperature incubator at 30°C for 48 hours to obtain fermented chicken liver protein hydrolysate (FCLPH). The fermentation product was heated at 85°C for 20 minutes to inactivate *Lactobacillus plantarum* and *Saccharomyces cerevisiae*, and this product was named the FCLPH group. The CLP group and CLPH group were treated the same as the FCLPH group except for the uninoculated bacterial solution (physiological saline was used instead). The CLP, CLPH, and FCLPH group samples were centrifuged at 8000 rpm for 10 minutes at 4°C. The supernatant was collected and used directly to determine the browning intensity. The precipitate was freeze-dried and stored at -20°C.

[0044] 3. Preparation of Maillard reaction products (i.e., chicken liver flavored seasoning)

[0045] Xylose (2%, w / v), L-cysteine ​​(1.5%, w / v), and thiamine hydrochloride (vitamin B1, 0.05%, w / v) were added to CLP, CLPH, and FCLPH solutions, respectively. The pH of each mixture was then adjusted to 7.0 using 0.5 M NaOH or 0.5 M HCl. The mixtures were reacted at 120 °C and 200 rpm / min for 90 min. After the reaction, the mixtures were immediately cooled rapidly in an ice-water bath. The resulting MRP solution was centrifuged at 8000 rpm for 10 min. The supernatant was collected and used directly for the determination of relevant indicators, while the precipitate was freeze-dried and stored at -20 °C for later use.

[0046] III. Determination of Physicochemical Properties

[0047] In this study, all test indicators were repeated at least three times, and results are expressed as mean ± standard deviation. Figures and tables were generated using software such as Origin 2021. Analysis of variance (ANOVA) was performed using SPSS Statistics 27.0 software, and significance was analyzed using the Duncan test in one-way ANOVA. p <0.05).

[0048] 1. Determination of pH value and browning intensity

[0049] The pH values ​​of CLP, CLPH, FCLPH, and their MRP supernatants were determined using a pH meter (FiveEasy Plus FE28, METTLER TOLEDO, Switzerland). The formation of intermediate and final products was assessed using a UV-Vis spectrophotometer (UV-3600i Plus; Shimadzu, China). Each supernatant was diluted 20-fold with deionized water, and the browning intensity of the MRPs was characterized by measuring the absorbance at 294 nm and 420 nm. The absorbance at 294 nm reflected the accumulation of colorless intermediates generated during the MR process, while the absorbance at 420 nm represented the accumulation of MR final products (such as melanoidins).

[0050] Under the same initial pH conditions, the pH value of the MR system is a key quantitative parameter for evaluating the reaction progress. For example... Figure 1 As shown, during enzymatic hydrolysis, proteins are hydrolyzed to produce acidic amino acids and release hydrogen ions (H+). + ), leading to system H + As concentration increased, pH decreased. The FCLPH group, in particular, experienced a further decrease in pH due to the additional influence of microbial metabolic acid production. The fermented hydrolysate group exhibited a lower final pH and higher total acid content, suggesting that these groups may possess richer flavor precursors. All MRPs showed a decreasing pH trend, likely due to the continued dehydration condensation of free amino groups from hydrolysates with the carbonyl groups of reducing sugars during MR, leading to a lower pH. Furthermore, the formation of substances such as formic acid, acetic acid, and free amino acids in MRPs may also contribute to the pH decrease. Clearly, the FCLPHM group of fermented chicken liver protein hydrolysate Maillard reaction products exhibited the lowest pH at the end of the reaction, indicating that fermentation treatment facilitated MR.

[0051] In MRP studies, absorbance measurements at 420 nm and 294 nm have been widely used as indicators of reaction extent. The absorbance at 294 nm primarily reflects the content of colorless or light-colored intermediates (such as aldehydes and ketones) generated in the early stages of MR, indicating the reaction stage; while the absorbance at 420 nm is positively correlated with the content of brown macromolecules such as melanoidins generated in the later stages, serving as a browning index to measure reaction intensity. Figure 2 The results showed that, compared with CLP, CLPH and FCLPH, their MRP had significantly higher absorbance at 420 nm and 294 nm. p<0.05). The increased absorbance indicates that Maillard reaction (MR) results in the formation of more intermediate products and browning compounds in the early to mid-stages. Furthermore, compared to the Maillard reaction products group (CLPM), the Maillard reaction products group (CLPHM) and FCLPHM of chicken liver protein hydrolysates showed higher absorbance, indicating an increased amount of intermediate products and browning compounds. This difference stems from the protein hydrolysis and fermentation processes. Trypsin hydrolyzes chicken liver protein, breaking down peptide chains, releasing free amino groups, and enhancing the interaction between amino groups and reducing sugars (such as xylose), thereby accelerating the MR process. Microbial fermentation further degrades animal protein, producing more small peptides, free amino acids, and other substances, providing a richer set of reaction sites. Therefore, FCLPH exhibits a higher degree of MR compared to CLPH.

[0052] 2. Particle size and free amino content

[0053] CLP, CLPH, and FCLPH, along with their MRPs, were prepared into 1 mg / ml solutions using deionized water. The particle size of the samples was determined using a nanoparticle size and Zeta potential analyzer (Zetasizer Nano-ZSE, Malvern Instruments Co., Ltd., Worcestershire, UK). Free amino groups were determined according to the OPA method to evaluate the degree of MR. Dissolve 40 mg OPA in 2 mL of methanol to ensure complete dissolution, then mix with 25 mL of 0.1 mol / L sodium tetraborate buffer (pH 9.5), 2.5 mL of 20% (w / v) SDS solution, and 100 μL of β-mercaptoethanol. Adjust the volume to 50 mL with ultrapure water. This reagent must be freshly prepared before each use. Adjust the volume of OPA reagent in the reaction system to 4 mL (mixed with 200 μL of sample), vortex for 15 seconds (25°C), incubate precisely in the dark for 2 minutes, and immediately measure the absorbance at 340 nm. Simultaneously, prepare a series of 0-3 mmol / L L-leucine standard solutions using the same matrix solvent.

[0054] The calibration equation is as follows: y = 0.0922x + 0.0541

[0055] Note: x and y represent the free amino content (mmol / L) and absorbance value, respectively.

[0056] Figure 3The results showed that MR significantly increased the average particle size of all samples, confirming the occurrence of the polymerization reaction. Specifically, the particle size of CLPM (6763 nm) increased by 528% compared to CLP (1077 nm), indicating that chicken liver protein formed large molecular aggregates during the reaction. Notably, the particle size of CLPHM (4001 nm) far exceeded that of CLPH (617 nm), with an increase of 548%, which is consistent with the theory that the high free amino content of CLPH promotes Maillard polymerization at the end. In contrast, FCLPHM (1431 nm) showed the smallest increase (213%). Furthermore, the basic particle size of FCLPH (458 nm) was significantly smaller than that of CLPH (617 nm), which may be due to the organic acid buffer system formed after adjusting the initial pH of MR. As MR progresses to the end, the acidic environment created by the organic acid may have inhibited the depth of the polymerization reaction to some extent.

[0057] like Figure 4 As shown, MR significantly reduced the free amino content in all samples, which is consistent with the mechanism by which amino groups participate in Maillard condensation as reactants. The free amino content in the FCLPH group (46.47 mM) was almost identical to that in the CLPH group (42.64 mM), indicating that fermentation did not significantly consume amino groups. The amino consumption in CLPHM and FCLPHM was more significant (reductions of 64.78% and 80.44%, respectively), further confirming that high initial amino content can significantly drive MR. However, the residual amino content in FCLPHM (16.41 mM) was significantly higher than that in CLPHM (9.09 mM). This phenomenon, seemingly contradicting the browning intensity result that "fermentation promotes MR," may stem from the production of a large number of active intermediates (α-dicarbonyl compounds, reductones, etc.) required for mid-to-late stage MR during fermentation. Therefore, although acid production during MR inhibits the first step of the carbonyl-amine condensation reaction, it still appears macroscopically as a promoting effect on MR, thus promoting the mid-to-late stage of MR reactions.

[0058] 3. Measurement of color difference

[0059] The color characteristics of each group of lyophilized samples were evaluated using a colorimeter (ZE7700, Nippon Denshoku Industries Co., Ltd., Japan). The values ​​of L*, a*, b*, and ΔE were also recorded. Five locations were randomly selected from each sample powder for the experiment. The total color difference was calculated using the following formula.

[0060] .

[0061] As shown in Table 1, the ΔE values ​​of CLP, CLPH, FCLPH, and their corresponding MRPs differed significantly, indicating that the synergistic effect of enzymatic hydrolysis, fermentation, and MR significantly altered the color characteristics of the products. Specifically, the FCLPH group showed a significant decrease in L* value and a significant increase in b* value after the MR reaction. This change is attributed to the degradation of Amadori products via furfural and reductone pathways during the MR process, as well as the subsequent accumulation of melanoidins and caramelization products. Compared to the CLPM and CLPHM groups, the FCLPHM group exhibited lower L* values ​​and higher a* and b* values. This suggests that fermentation treatment may promote glycosylation by releasing free amino groups or MR intermediates, thereby accelerating the MR reaction process and leading to the formation of more brown substances.

[0062] Table 1. Effects of compound fermentation treatment on the color changes of chicken liver protein hydrolysate and its MR products.

[0063]

[0064] Note: Results are expressed as mean ± standard error (n=3). Different letters a–d in the same column indicate significant differences in the mean. p <0.05)

[0065] IV. Determination of Flavor Compounds

[0066] 1. Measurement of electronic nose

[0067] The odor characteristics of the samples were analyzed using an e-nose odor analyzer (Bosin Industrial Development Co., Ltd., Shanghai, China). The core of this instrument is an array of 18 metal-oxide-semiconductor (MOS) sensors (the sensitive substances for each sensor are shown in Table S1), which can specifically identify different types of volatile organic compounds. 10 mL of sample solution was placed into a 40 mL sealed headspace vial and equilibrated in a 60 °C water bath for 20 min to allow for the full release of volatile components into the headspace vial. The volatile compound test lasted for 60 s, and the acquired raw signal data was processed and analyzed using the accompanying electronic nose software.

[0068] Table 2 Electronic Nose Sensor Configuration Table

[0069]

[0070] Electronic noses can identify and analyze the type, composition, and concentration of gases, thereby providing a comprehensive characterization of the odor profile of a sample. Electronic nose analysis ( Figure 5 This indicates that there are significant differences in the odor characteristics of samples from different treatment groups. p<0.05). After MR treatment, the response values ​​of sensors S4, S16 (sulfur-containing compounds), S5 (nitrogen heterocyclic compounds: such as pyrazines / furans), S6 (aldehydes and ketones), and S9 (alcohols and ketones) were significantly improved. This indicates that these substances are the main contributing components of the sample odor, a result consistent with the formation mechanism of nitrogen-containing heterocyclic compounds and carbonyl-amino reaction products during heat treatment. The synergistic effect of enzymatic hydrolysis, fermentation, and MR enhanced the release of volatile organic compounds (VOCs). Compared to their individual precursors, the overall odor response values ​​of MRPs were significantly increased, with the FCLPHM group achieving the highest overall response value.

[0071] 2. Measurement of electronic tongue

[0072] Taste characteristics of samples were analyzed using an e-tongue instrument (Bosin Industrial Development Co., Ltd., Shanghai, China). This instrument is equipped with a multi-sensor array containing six metal electrodes (platinum, gold, palladium, titanium, tungsten, and silver). Before testing, the probes were cleaned with ultrapure water for 2 minutes. The signal amplification factor was set to 100x, the signal acquisition range was [−2, 2], and the sensor response time was 180 s. During analysis, the electrodes were immersed in 30 mL of sample solution for 30 s, and the system recorded the response values ​​of the six sensors. The intensity value of each taste was calculated using multivariate analysis. All data were acquired and analyzed using the accompanying e-tongue software.

[0073] Electronic tongues can identify and analyze the taste, composition, and pH of liquids, thereby characterizing the taste features of a sample. Electronic tongue analysis ( Figure 6 This indicates that there are significant differences among the groups in terms of the intensity of astringency, sourness, sweetness, and umami. p <0.05), while the difference in bitterness and saltiness intensity was not significant ( p >0.05). The overall response value of CLP was low, which is related to its large molecular weight and difficulty in binding to taste receptors. CLPH showed the highest response intensity for bitterness and astringency, presumably due to the enzymatic hydrolysis producing hydrophobic amino acids and small peptides, leading to a decrease in taste perception. FCLPH, after fermentation, showed a significant increase in acidity, attributed to lactic acid produced by Lactobacillus plantarum fermentation; at the same time, its bitterness and astringency intensity decreased significantly, possibly due to the masking effect of organic acids on bitterness or the consumption of bitter substances by microbial metabolism.

[0074] Compared to their precursors, MRPs showed significantly improved taste acceptability. CLPHM exhibited the highest sweetness response value, attributed to the abundant free amino acids and small amount of reducing sugars released during enzymatic hydrolysis, which promoted the formation of a large number of sweet furanone compounds. This corresponds to the high 2-pentylfuran content in CLPHM observed in GC-MS analysis. FCLPHM, on the other hand, possessed the highest umami response value, possibly due to the ample provision of umami amino acid precursors by enzymatic hydrolysis, while fermentation contributed nucleotides with synergistic umami effects.

[0075] 3. Determination of the composition of free amino acids (FAAs)

[0076] The content of free amino acids was determined using a liquid chromatography system equipped with a UV detector (LC-2010C, Shimadzu Corporation, Kyoto, Japan). 0.1 g of sample was accurately weighed, and 2 mL of an aqueous solution containing α-aminobutyric acid (0.5 mg / mL) as an internal standard was added. The volume was adjusted to 25 mL with water, and the sample was extracted by sonication for 30 min, followed by centrifugation (5 min). 200 µL of the supernatant was collected, and 1 mL of pH 9.0 sodium carbonate buffer and 200 µL of 3% 2,4-dinitrochlorobenzene (CDNB) acetonitrile derivatizing agent were added. Derivatization was performed at 95 °C in the dark for 150 min. After cooling, 200 µL of 10% glacial acetic acid was added for neutralization, and the sample was filtered through a 0.22 µm organic filter before injection. Separation was performed using a CAPCELL PAK C18 MGII column (250 × 4.6 mm, 5 µm; Shiseido Co., Ltd., Tokyo, Japan) at 40 °C. The mobile phase consisted of acetonitrile (A) and triethylamine-containing acetate-sodium acetate buffer (B) in gradient elution mode at a flow rate of 1.0 mL / min. The detection wavelength was 360 nm. Quantification was performed using the internal standard method.

[0077] By measuring the changes in the composition of free amino acids in samples before and after MR (metabolism), the degree to which amino acids participate in the reaction and their impact on flavor can be reflected. For example... Figure 7 As shown, due to the interaction between the carbonyl group of reducing sugar and the free amino group, the total free amino acid content in all MRPs showed a decreasing trend. The total free amino acid content in the CLPM, CLPHM, and FCLPHM groups decreased to 90.22%, 86.24%, and 79.65% of the pre-reaction levels, respectively. Notably, after the MR reaction, the free amino acid content in the FCLPH group significantly decreased from 44.37 mg / g to 35.34 mg / g, indicating that fermentation treatment promoted the participation of CLPH in the MR process.

[0078] The content of bitter amino acids decreased in all MRPs, with particularly significant reductions in the CLPHM and FCLPHM groups, at 40.98% and 51.90%, respectively. The reduction of Leu, Ile, and Arg played a dominant role in this process (for example, in FCLPHM, these three decreased by 79.56%, 68.57%, and 65.94%, respectively). Given the strong bitterness of Leu and Ile, their significant reduction is crucial for improving the overall bitterness of the MRPs. Meanwhile, the content of sweet amino acids increased significantly in both the CLPHM and FCLPHM groups, with increases of 33.98% and 11.18%, respectively. The increase in Gly, with its strong sweetness, was particularly significant, reaching 50.64% and 73.77%. Furthermore, unlike other groups, the content of umami amino acids (Asp and Glu) in the FCLPHM group increased significantly by 34.39%, which corroborates the enhanced umami results detected by the electronic tongue.

[0079] 4. GC-MS Measurement

[0080] VOCs in the sample were analyzed using headspace solid-phase microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS, Agilent GC-MS system, model: 8890 GC and 5977B MSD, China). The specific procedure was as follows: 3.0 g of sample was accurately weighed into a 20 mL headspace sample vial, and 10 μL of 10 μL headspace microextraction gas chromatography-mass spectrometry (HS-SPME-GC-MS, Agilent GC-MS system, model: 8890 GC and 5977B MSD, China) was added. 4 A diluted 2,4,6-trimethylpyridine (TMP) solution was used as an internal standard. After aging the SPME extraction fiber needle, it was inserted into the headspace vial, and the vial was heated in a 60 °C water bath for 30 min for enrichment extraction. After extraction, the needle was removed and immediately inserted into the GC injection port, and desorption was performed at 250 °C for 10 min. Gas chromatography conditions were as follows: an HP-5MS UI column (30 m × 250 μm·d. × 0.25 μm film thickness), a split ratio of 5:1, and a temperature program of initial column temperature of 40 °C held for 3 min, followed by ramping to 250 °C at a rate of 5 °C / min and holding for 12 min, for a total run time of 57 min. The mass spectrometry conditions were: electron impact ionization (EI) source, ion source temperature 230 °C, MS quadrupole temperature 150 °C, solvent delay time 1 min, and scanning mass range 35–400 m / z.

[0081] A total of 106 volatile compounds were identified from CLP, CLPH, FCLPH and their corresponding MRPs using GC-MS, including aldehydes (21), heterocyclic compounds (20), ketones (19), hydrocarbons (19), alcohols (13), esters (4), acids (3) and others (7), with a total concentration ranging from 429.53 μg / kg to 3518.96 μg / kg (Table 3).

[0082] Table 3. Content of volatile compounds in different MR precursors and their corresponding MRPs

[0083] ;

[0084] ;

[0085] ;

[0086] ;

[0087] ;

[0088] ;

[0089] ;

[0090] Note: Results are expressed as mean ± standard error (n=3). A significant difference in the mean is indicated by different letters a–d within the same row. p < 0.05).

[0091] CLP exhibits fewer VOC types and lower concentrations, which is related to its larger molecular weight. CLPH and FCLPH have similar VOC compositions, primarily rich in small-molecule aldehydes and ketones. In contrast, the three MR products (CLPM, CLPHM, and FCLPHM) show a richer variety and higher concentration of VOCs. Among them, heterocyclic compounds such as pyrazines and furans, which possess aromas of roasted meat, nuts, and cooked meat, are characteristic flavor substances of MR.

[0092] Aldehydes are the main products of lipid degradation and Strecker degradation of amino acids. In addition, carbonyl compounds derived from protein oxidation can also produce Strecker aldehydes, primarily imparting floral, fruity, and fatty aromas to precursors and their MRPs. Pentanal, hexanal, nonanal, and benzaldehyde were present in high amounts in CLPH and FCLPH, mainly due to the oxidation of small amounts of fats in CLP. The types and amounts of aldehydes in each group of MRPs further increased. For example, methylthiopropional (imparting roasted nut and onion spiciness) and furfural (presenting roasted almond and caramel sweetness) were present in the FCLPHM group at levels as high as 153.78 μg / kg and 159.34 μg / kg, respectively. Notably, compared to the FCLPHM group, higher levels of octanal (fatty, grassy), hexanal (grassy, ​​oily, sweaty), and 1-penten-3-one (fishy, ​​spicy) were detected in CLPM and CLPHM. These substances, at higher concentrations, may produce unpleasant off-flavors, affecting the flavor quality of the MRPs.

[0093] 5. Calculation of OAV value

[0094] Odor Activity Value (OAV) represents the contribution of volatile compounds to flavor. It is calculated by dividing the concentration of a volatile compound by its odor threshold. Compounds with a ratio value ≥ 1 are considered to be the cause of aroma, and their OAV is proportional to their contribution to aroma characteristics.

[0095] Table 4. Odor Activity Values ​​(OAV) of different MR precursors and their corresponding MRPs volatile compounds

[0096] ;

[0097] ;

[0098] Note: "–" indicates that no detection was performed.

[0099] Figure 8This paper presents the Pearson correlation analysis results of 35 key volatile organic compounds with odor activity values ​​(OAV) > 1 with the electronic nose sensor and electronic tongue taste. Strong correlations were generally observed among the response values ​​of the various electronic nose sensors, reflecting the cross-sensitivity characteristics of the electronic nose sensors. This means that a single sensor typically responds to a class of compounds with similar chemical properties, rather than targeting only a single substance. Therefore, further analysis using GC-MS results is necessary. The analysis results confirmed the effectiveness of this binding. For example, 3-methylbutyric acid, 2,6-dimethylpyrazine, and 2-ethyl-2-hexenal showed a significant negative correlation with sensor S9 (alkanes, alcohols, and ketones); 2,3-dimethylpyrazine, 2,5-dimethylpyrazine, and pyrazine showed a significant positive correlation with sensor S5 (nitrogen oxides); while 2-acetylthiazole, thiazole, methylthiopropional, and methanethiol showed a strong correlation with sensors S4 and S16 (both sensitive to sulfides). These results effectively validated the reliability of the electronic nose data and showed good consistency with the changes in key flavor compounds detected by GC-MS.

[0100] Regarding taste, the analysis revealed specific associations between VOCs and the electronic tongue response. Acetic acid and 3-methylbutyric acid showed a significant positive correlation with the electronic tongue sour taste sensor response values, consistent with their taste properties. More importantly, the analysis found a significant positive correlation between 2,5-dimethylpyrazine (pyrazines) and 2-acetylthiazole (thiazoles) and the electronic tongue umami sensor response values, supporting the view that MR has an umami-enhancing effect. These compounds with potential umami-enhancing effects were present in significantly higher amounts in MRPs (especially FCLPHMs) than in their precursors. This, combined with changes in the umami amino acid composition of MRPs (… Figure 7 ) and the enhanced umami intensity detected by electronic tongue ( Figure 6 Together, they elucidated why the FCLPHM sample exhibited the best umami characteristics.

[0101] In summary, this invention innovatively combines the co-fermentation of isolated *Lactobacillus plantarum* and *Saccharomyces cerevisiae* with MR (methanethiamine oxidase), significantly improving the flavor and structural characteristics of CLPH (Clearly Chlorinated Protein). The dual-strain co-fermentation, through deep protein hydrolysis, increases the free amino acid content in CLPH, while microbial metabolism produces MR intermediate active products; both optimize the MR precursors. A total of 106 volatile flavor compounds were identified after MR, of which 35 had an OAV > 1. The content of key flavor compounds (methylthiopropionaldehyde, pyrazine, 2,3-butanedione, etc.) in FCLPHM was significantly increased, mainly contributing to meat, nut, and creamy aromas; unpleasant odor compounds (such as 1-penten-3-one) were significantly reduced, mainly contributing to fishy odors. Free amino acid composition analysis showed that fermentation treatment reduced the level of bitter amino acids and enriched umami and sweet amino acid components. Electronic nose and electronic tongue analysis confirmed its rich flavor layers and prominent umami. UV, fluorescence, and infrared spectroscopy characterized the molecular interaction mechanism of precursor substances and the structural changes of corresponding MRPs during the MR process. This study provides theoretical support for the development of novel animal by-product seasonings and is expected to promote the high-value utilization of chicken liver by-products.

[0102] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The application of a compound microbial agent in the preparation of chicken liver flavored seasoning, characterized in that: The compound microbial agent is composed of Lactobacillus plantarum YR07 ( Lactiplantibacillus plantarum ) and brewer's yeast HQSAJ-001-24 ( Saccharomyces cerevisiae Activate and culture separately until the viable bacterial count is ≥ 1×10⁻⁶. 7 CFU / mL, mixed in a volume ratio of 1:1-3; The Lactobacillus plantarum YR07 ( Lactiplantibacillus plantarum It is deposited at the China Center for Type Culture Collection on August 18, 2022, with the biological accession number CCTCC NO:M20221303; The brewing yeast HQSAJ-001-24 ( Saccharomyces cerevisiae It is deposited at the China General Microbiological Culture Collection Center (CGMCC) on September 16, 2025, with the biological accession number CGMCC No. 35983.

2. The application of the compound microbial agent according to claim 1 in the preparation of chicken liver flavored seasoning, characterized in that, Includes the following steps: (1) Preparation of chicken liver protein hydrolysate: Fresh or frozen chicken liver is used as raw material. After washing, removing blood vessels and bile ducts, homogenizing, heating to inactivate enzymes, then defatting, centrifuging and taking the precipitate to obtain chicken liver protein. The chicken liver protein is redissolved in water, the pH is adjusted to 7.5-8.5, trypsin is added for hydrolysis, and then the enzyme is inactivated to obtain chicken liver protein hydrolysate. (2) Fermentation of compound microbial agent: The compound microbial agent described in claim 1 is inoculated into the chicken liver protein hydrolysate obtained in step (1) at a total inoculation amount of 2%, and glucose is added at the same time to adjust the initial pH to 6.0-7.

0. Fermentation is carried out, and then the fermentation is terminated by heat treatment to obtain fermented chicken liver protein hydrolysate. (3) Maillard reaction flavor enhancement: Xylose, L-cysteine ​​hydrochloride and thiamine hydrochloride were added sequentially to the fermented modified chicken liver protein hydrolysate obtained in step (2), and the pH was adjusted to 6.5-7.5 after stirring evenly. The mixture was placed in a reaction vessel and stirred to react. After the reaction was completed, the mixture was immediately cooled in an ice bath to terminate the reaction. (4) Product post-processing and preparation: Centrifuge the Maillard reaction product obtained in step (3), collect the supernatant, and then spray dry it to obtain chicken liver flavor seasoning powder product.

3. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (1), the enzyme inactivation is performed by heating at 85-90°C for 15-20 minutes; the defatting is performed by using 50-60% isopropanol at a material-to-liquid ratio of 1:4–1:

6.

4. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (1), the amount of trypsin added is 1% of the substrate mass, the hydrolysis conditions are hydrolysis at 40 °C for 2-4 hours, and the enzyme is inactivated at 85-90 °C for 10-15 minutes after hydrolysis.

5. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (2), the amount of glucose added is 5%, the fermentation conditions are fermentation at 30℃ for 36-48 hours, and the heat treatment is to terminate the fermentation by heat treatment at 85-90℃ for 10-15 minutes.

6. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (3), the amount of xylose added is 1.5-2.5% of the dry basis mass of the hydrolysate, the amount of L-cysteine ​​hydrochloride added is 0.5-1.5% of the dry basis mass of the hydrolysate, and the amount of thiamine hydrochloride added is 0.03-0.07% of the dry basis mass of the hydrolysate.

7. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (3), the reaction conditions are 115-125℃ and 150-250 rpm for 60-120 minutes.

8. The application of the compound microbial agent according to claim 2 in the preparation of chicken liver flavored seasoning, characterized in that: In step (4), the centrifugation conditions are 8000 rpm for 10-15 minutes, the inlet air temperature of the spray drying is 170-190℃, and the outlet air temperature is 80-90℃.

9. The application of the compound microbial agent according to claim 1 in the preparation of chicken liver flavored seasoning, characterized in that: The chicken liver flavor seasoning contains a total protein content of ≥60%, a total free amino acid content of ≥35 mg / g, of which umami amino acids account for ≥25% and sweet amino acids account for ≥30%; a total pyrazine compound content of ≥80 μg / kg, a total furan compound content of ≥100 μg / kg, and a sulfur-containing compound content of ≥120 μg / kg.

Images