A strain of Lactobacillus plantarum capable of degrading low molecular weight aldehydes and its combination and application in degradation of duck liver odor

Abstract

The invention discloses a Lactobacillus plantarum strain capable of degrading low-molecular aldehydes, a combination thereof, and application of the strain in degrading the fishy smell of duck liver. The strain is characterized in that the strain is an LPC01 strain with a deposit number of CGMCC No. 32002, and the addition amount of the Lactobacillus plantarum suspension to duck liver pâté is 0.5-1.5vt%. The invention further provides an application of the Lactobacillus plantarum combination consisting of a Lactobacillus plantarum suspension and a yeast suspension in equal volumes in degrading the fishy smell of duck liver. The combination comprises the steps of inoculating the yeast suspension into the duck liver pâté for fermentation, and then inoculating the Lactobacillus plantarum suspension for fermentation. The invention has the advantages that, during the fermentation process of the duck liver, the total aldehyde degradation rate of the strain is 63.8%, and under the condition of co-culturing with the yeast, the degradation rate is increased to 97.8%, showing a significant fishy smell removal effect.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to a plant lactobacillus strain capable of degrading low-molecular aldehydes, a combination thereof, and an application thereof in degrading the fishy smell of duck liver. Background Art

[0002] A prominent fishy odor is a common flavor deterioration in animal-derived foods and their by-products (blood, liver, kidney, intestines, etc.), as well as plant-derived foods such as soybeans and kelp. This is particularly true of by-products generated during livestock and poultry meat processing, such as non-foie gras-grade waterfowl livers like duck and goose liver. Although rich in nutrients such as protein, vitamins, minerals, and unsaturated fatty acids, these by-products possess high economic and nutritional value. However, during intensive processing, the high content and volatility of fishy odor compounds present a major challenge in developing value-added products. Fatty aldehydes are the primary source of fishy odor in animal and plant foods, encompassing saturated and unsaturated aldehydes such as enals and dienals. Aldehydes are biosynthesized through metabolism in animals and plants and are also formed as products of lipid oxidation and Strecker degradation of amino acids during storage. Fatty aldehydes have a low fishy odor threshold, indicating that even at low concentrations, they can significantly affect the sensory properties of related foods. Therefore, eliminating or mitigating the fishy odor deterioration caused by fatty aldehydes is a key issue that needs to be addressed in order to achieve intensive processing of livestock and poultry liver products.

[0003] At present, there are three main types of deodorization technologies for waterfowl liver: physical, chemical, and biological methods. Physical deodorization methods such as pickling, filtering, and elution are simpler to operate and less expensive than chemical and biological methods. They are the most commonly used deodorization methods for simple processing of livestock and poultry by-products. However, physical methods are usually unable to fundamentally remove fishy substances, and the deodorization efficiency fluctuates greatly and is affected by factors such as the form of raw materials and the region where the product is consumed. Chemical methods use chemical substances to block the generation pathways of fishy compounds during processing to achieve the effect of removing the fishy smell of food. However, they often cause food safety problems due to the failure to remove residues after the addition of chemical substances. As an efficient and safe method, biological deodorization methods have been widely used in recent years to remove odors from food. Biological deodorization that relies on enzyme treatment and microbial fermentation is a potential important means to improve the flavor of meat and aquatic products. However, the lack of specific enzyme preparations and efficient strains, as well as the insufficient development of deodorization technologies and optimized processes based on product characteristics, significantly limits the industrial application of this method. Summary of the Invention

[0004] The technical problem to be solved by the present invention is to provide a strain of Lactobacillus plantarum that can efficiently degrade low-molecular aldehydes such as (E)-2-hexenal, (E,E)-2,4-hexadienal, heptanal, hexanal, octanal and benzaldehyde, as well as a combination thereof and an application thereof in degrading the fishy smell of duck liver.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a strain of Lactobacillus plantarum that degrades low molecular weight aldehydes, the strain being LPC01, classified and named Lactobacillus plantarum ( Lactobacillus plantarum ), deposited with the China General Microbiology Center under the China Culture Collection Administration on September 19, 2024, with the accession number CGMCC No. 32002. Lactobacillus plantarum LPC01 has excellent degradation capacity for low-molecular-weight aldehydes, including (E)-2-hexenal, (E,E)-2,4-hexadienal, heptanal, hexanal, octanal, and benzaldehyde.

[0006] The present invention also provides the application of the plant lactobacillus in degrading the fishy smell of duck liver, wherein the amount of the plant lactobacillus suspension added to the duck liver paste is 0.5-1.5vt%, and the OD value of the plant lactobacillus suspension is 0.5-1.5vt%. 600 The value is 0.5-2.

[0007] The present invention also provides a plant lactobacillus combination for degrading low molecular weight aldehydes. The combination is composed of a plant lactobacillus suspension and a yeast suspension of equal volume. The OD of the plant lactobacillus suspension is 600 The value is 0.5-2, and the OD of the yeast suspension is 600 The value is 0.5-2.

[0008] Furthermore, the plant lactobacillus is a strain LPC01, which is classified as plant lactobacillus ( Lactobacillus plantarum ), deposited in the General Microbiology Center of China Culture Collection Administration on September 19, 2024, with the deposit number CGMCC No.32002.

[0009] The present invention also provides an application of the above-mentioned Lactobacillus plantarum combination in degrading the fishy smell of duck liver. The yeast suspension is inoculated into duck liver paste and fermented at 25-35°C for 0.5-1.5 hours. The Lactobacillus plantarum suspension is then inoculated and fermented for 1.5-2.5 hours. The inoculation volume ratio of the yeast suspension to the Lactobacillus plantarum suspension is 1:1, and the total inoculation amount is 0.5-1.5vt%.

[0010] Furthermore, the OD of the Lactobacillus plantarum suspension is 600 The value is 0.5-2, and the OD of the yeast suspension is 600 The value is 0.5-2.

[0011] Compared with existing technologies, the present invention offers advantages in that it discloses a strain of Lactobacillus plantarum that degrades low-molecular-weight aldehydes, as well as combinations thereof, and their application in degrading the fishy odor of duck liver. Strain LPC01 was isolated from fermented kimchi and can degrade hexanal, octanal, (E)-2-hexenal, (E,E)-2,4-hexadienal, and heptanal by 48% to 100% within 72 hours. The large number of reductive dehydrogenases and hydrogen-donating intermediate metabolites produced by Lactobacillus plantarum during fermentation metabolism can potentially promote the conversion of volatile, fishy, ​​small-molecule fatty aldehyde compounds in foods into odorless or low-threshold compounds. Furthermore, Lactobacillus plantarum exhibits high substrate utilization efficiency and a strong ability to form characteristic volatile flavor compounds such as acids, esters, and alcohols. During fermentation, it produces lactic acid and other organic acids, lowering the pH, which helps inhibit the growth of undesirable microorganisms and improves the overall flavor of the food. During duck liver fermentation, the strain's total aldehyde degradation rate was 63.8%. In co-culture with yeast, the degradation rate increased to 97.8%, demonstrating significant fishy odor removal and promising application prospects. Therefore, Lactobacillus plantarum LPC01 and its combination with yeast have significant application prospects in removing fishy odor from livestock and poultry livers.

[0012] The above-mentioned Lactobacillus plantarum is a strain LPC01, which is classified and named as Lactobacillus plantarum ( Lactobacillus plantarum ), deposited in the General Microbiology Center of China Culture Collection of Microorganisms on September 19, 2024, with the deposit number CGMCC No. 32002, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results of the determination of the degradation rate of hexanal by lactic acid bacteria;

[0014] Figure 2 This is the result of the determination of the degradation rate of heptanal by lactic acid bacteria;

[0015] Figure 3 The results of the determination of the degradation rate of octanal by lactic acid bacteria;

[0016] Figure 4 The results of the determination of the degradation rate of (E)-2-hexenal by lactic acid bacteria;

[0017] Figure 5 The results of the degradation rate of (E,E)-2,4-hexadienal by lactic acid bacteria are shown in Table 1.

[0018] Figure 6 This is the result of the determination of the degradation rate of benzaldehyde by lactic acid bacteria;

[0019] Figure 7is the colony morphology of Lactobacillus plantarum LPC01;

[0020] Figure 8 is the Gram staining result of Lactobacillus plantarum LPC01;

[0021] Figure 9 This is the SEM morphology observation of Lactobacillus plantarum LPC01;

[0022] Figure 10 This is the phylogenetic tree result of Lactobacillus plantarum LPC01;

[0023] Figure 11 is the growth curve of Lactobacillus plantarum LPC01;

[0024] Figure 12 is a graph showing the acid production capacity of Lactobacillus plantarum LPC01;

[0025] Figure 13 The results of the aldehyde degradation rate determination after co-cultivation of Lactobacillus plantarum LPC01 and yeast;

[0026] Figure 14 This is the result of acetaldehyde dehydrogenase activity after co-cultivation of Lactobacillus plantarum LPC01 and yeast;

[0027] Figure 15 This is the result of alcohol dehydrogenase activity after co-cultivation of Lactobacillus plantarum LPC01 and yeast;

[0028] Figure 16 This is the result of intracellular protease activity after co-cultivation of Lactobacillus plantarum LPC01 and yeast;

[0029] Figure 17 This is the result of extracellular protease activity after co-cultivation of Lactobacillus plantarum LPC01 and yeast;

[0030] Figure 18 This is the heat map result of the volatile flavor compound content of duck liver after co-cultivation of Lactobacillus plantarum strain LPC01 and yeast;

[0031] Figure 19 The results show the changes in the content of volatile flavor compounds of different chemical categories in duck liver after co-cultivation of Lactobacillus plantarum strain LPC01 and yeast.

[0032] Figure 20 These are the results of the effects of Lactobacillus plantarum strain LPC01 and its co-culture on the content of aldehydes in duck liver. DETAILED DESCRIPTION

[0033] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0034] Specific embodiment 1, screening and identification of target strains, including the following steps:

[0035] Step 1. Initial screening of target strains: Fermented kimchi samples were serially diluted and plated on MRS agar containing 6 wt% bromocresol purple in triplicate. Each plate was incubated at 37°C for 1-2 days, and the growth of colonies on the plates was observed. Colonies with different morphologies were streaked and isolated repeatedly until three single strains were obtained. These strains were tentatively named LPC01, LPC02, and LPC03. The MRS agar medium formulation was as follows: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, agar 14.0 g / L, Tween 80 1.0 g / L, pH 6.5 ± 0.2, 25°C.

[0036] Step 2: Rescreening of target strains: strains LPC01, LPC02, and LPC03 were inoculated into MRS broth, activated at 37°C and 180 rpm for 8-12 h, and passaged twice. 2 mL of culture medium was centrifuged at 4°C and 10,000 rpm for 5 min, the supernatant was discarded, and the cells were washed twice with PBS buffer and the OD values ​​were calculated. 600 The value was adjusted to 0.8-1. The activated culture medium was transferred to MRS broth containing 500 ppm of aldehydes (hexanal, octanal, benzaldehyde, (E)-2-hexenal, (E,E)-2,4-hexadienal, and nonanal at 500 ppm) at a volume percentage of 1%. The culture was cultured at 37°C and 180 rpm for 0-3 days. The aldehyde content in the culture medium was measured and the aldehyde degradation rate was calculated. Three replicates were performed for each group. MRS broth containing 500 ppm of aldehydes (uninoculated) served as the control group. The MRS broth medium formula is as follows: peptone 10.0 g / L, beef extract powder 8.0 g / L, yeast extract powder 4.0 g / L, glucose 20.0 g / L, dipotassium hydrogen phosphate 2.0 g / L, diammonium hydrogen citrate 2.0 g / L, sodium acetate 5.0 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.04 g / L, Tween 80 1.0 g / L, pH 5.7 ± 0.2 at 25°C.

[0037] The fatty aldehyde degradation rate was determined using the direct water injection method. The culture broth was centrifuged at 4°C and 10,000 rpm for 5 minutes. The supernatant was collected, filtered through a 0.22 µm water filter, and transferred to a brown vial for analysis. Low-molecular-weight fatty aldehydes were determined using an Agilent 7890B gas chromatograph equipped with a DB-WAX column and an FID detector. The inlet temperature was set at 200°C with a split ratio of 10:1. The column temperature was initially set at 40°C, then increased to 200°C at a rate of 5°C / min and held for 2 minutes. The detector temperature was set at 250°C, the injection volume was 0.4 µL, and the nitrogen flow rate was 30 mL / min. The aldehyde degradation rate formula is: Aldehyde degradation rate (%) = (aldehyde content before fermentation - aldehyde content after fermentation) / aldehyde content before fermentation × 100%. The degradation rates of hexanal, heptanal, octanal, (E)-2-hexenal, (E,E)-2,4-hexadienal and benzaldehyde were used as evaluation indicators, and the results were as follows. Figure 1 、 Figure 2 、 Figure 3 、 Figure 4 、 Figure 5 and Figure 6 As shown, the strain LPC01 with outstanding fatty aldehyde degradation performance was finally screened out from the candidate strains.

[0038] Step 3: Identification of target strains

[0039] 1. Morphological analysis and physiological and biochemical identification: The strain LPC01 was cultured on MRS agar plate at 37℃ for 24-48h to grow single colonies. Figure 7 As shown, its morphology is milky white, small and dense, opaque, with regular edges and a smooth surface. Strain LPC01 was cultured on MRS agar medium for 24 hours, placed on a glass slide with sterile water, and a small amount of bacteria was picked up with a needle and evenly spread in the water. After fixation, Gram staining was performed and the morphology of the strain was observed under a 100× optical microscope using an oil lens. The results are shown in Figure 1. Figure 8 As shown, the Gram staining result is purple, indicating that LPC01 is a Gram-positive bacterium. Figure 9 The following is a scanning electron micrograph of strain LPC01, which is rod-shaped. The physiological and biochemical characteristics of strain LPC01 are shown in Table 1.

[0040] Table 1 Physiological and biochemical characteristics of strain LPC01

[0041]

[0042] Based on the morphological and physiological and biochemical identification results, the LPC01 strain was preliminarily identified as a Lactobacillus genus.

[0043]

[0044] The sequencing results were compared in NCBI, and the 16S rDNA sequence of strain LPC01 was compared with Lactobacillus plantarum With 100% similarity, combined with physiological and biochemical data analysis, the strain LPC01 was determined to be Lactobacillus plantarum. The phylogenetic tree is as follows Figure 10 As shown. The strain has been deposited in the General Microbiology Center of China Culture Collection Administration of Microorganisms and is classified as Lactobacillus plantarum ( Lactobacillus plantarum ), LPC01 strain, the deposit number is CGMCC No. 32002, the deposit date is September 19, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, Institute of Microbiology, Chinese Academy of Sciences.

[0045] 3. Strain growth curve and acid production experiment: The screened LPC01 strain was inoculated into MRS broth medium and cultured at 37°C and 180 rpm for 8-12 hours. After two passages, the strain was centrifuged at 4°C and 10,000 rpm for 5 minutes, the supernatant was discarded, and the strain was washed twice with PBS buffer and the OD value was calculated. 600 The value was adjusted to 1. Take 1 mL of Lactobacillus plantarum LPC01 suspension and inoculate it into MRS broth medium. Culture it for 32 h and measure the OD value every 2 h. 600 Measure the pH value every 2 hours. Figure 11 As shown in the figure, strain LPC01 is in the retardation phase from 0 to 4 hours, with slow growth; it enters the rapid growth phase from 4 to 12 hours, enters the stable growth phase from 12 to 24 hours, and enters the decay phase after 24 hours. Figure 12 As shown in the figure, the pH of strain LPC01 dropped below 5.00 after culturing for 6 h, indicating that the degrading bacteria had a good acid production ability.

[0046] Specific Example 2: Co-culture of Lactobacillus plantarum LPC01 and yeast to degrade aldehyde and determine enzyme activity.

[0047] 1. Preparation of fermentation broth:

[0048] The plant lactobacillus LPC01 obtained by screening in the specific embodiment 1 was cultured at 37°C and 180 rpm for 8-12 hours to obtain a lactic acid bacteria suspension. Rhodotorula mucilaginosa , abbreviated as Yeast) were cultured at 30°C, 180 rpm for 12-48 h, subcultured twice, centrifuged at 4°C, 10,000 rpm / min for 5 min, the supernatant was discarded, and the cells were washed twice with PBS buffer and the OD 600 The value was adjusted to 0.8 to obtain a yeast suspension.

[0049] The lactic acid bacteria suspension and the yeast suspension were mixed in a volume ratio of 1:1 and inoculated into MRS broth containing low-molecular-weight fatty aldehydes (including hexanal, octanal, benzaldehyde, (E)-2-hexenal, (E,E)-2,4-hexadienal and heptanal, all at a concentration of 500 ppm) at a volume percentage of 1vt%. The culture was incubated at 30°C and 180 rpm for 24 h. The resulting fermentation broth was designated as the LPC01+Yeast group.

[0050] The lactic acid bacteria suspension was inoculated into MRS broth containing 500 ppm of aldehydes (hexanal, octanal, benzaldehyde, (E)-2-hexenal, (E,E)-2,4-hexadienal, and heptanal, at a concentration of 500 ppm, respectively). The culture was incubated at 30°C and 180 rpm for 24 h. The resulting fermentation broth was designated as the LPC01 group.

[0051] 2. Determination of aldehyde degradation rate: The method for determining aldehyde degradation rate is the same as step 2 in the above specific embodiment 1. The determination results are as follows: Figure 13 As shown in the results, the degradation rates of hexanal, octanal, (E,E)-2,4-hexadienal, (E)-2-hexenal, heptanal, and benzaldehyde in the LPC01 group were 78.4%, 66.9%, 62.1%, 76.6%, 75.6%, and 36.1%, respectively. The degradation rates of hexanal, octanal, (E,E)-2,4-hexadienal, (E)-2-hexenal, heptanal, and benzaldehyde in the LPC01+Yeast group were 83.2%, 73.4%, 72.0%, 84.7%, 82.1%, and 43.4%, respectively. Compared with the LPC01 group, the degradation rates of hexanal, octanal, (E,E)-2,4-hexadienal, (E)-2-hexenal, heptanal, and benzaldehyde increased by 4.7%, 6.5%, 9.9%, 8.1%, 6.6%, and 7.3%, respectively. This result indicates that co-culturing strain LPC01 with yeast significantly enhances the ability to degrade aldehydes. The co-culture results of lactic acid bacteria and yeast demonstrate metabolic complementarity between the two, which helps promote metabolic activity in the aldehyde conversion pathway. Lactic acid, as an organic acid, not only effectively lowers the pH of the culture medium, creating a moderately acidic environment, but also promotes the degradation of aldehydes by regulating yeast cellular metabolism, enhancing the expression and activity of related enzymes and degradative enzymes. Furthermore, as a substrate in the yeast metabolic pathway, lactic acid participates in the degradation process, further promoting the biotransformation of aldehydes and thereby increasing the efficiency of aldehyde degradation.

[0052] 3. Assay of acetaldehyde dehydrogenase activity

[0053] Preparation of crude enzyme solution: 50 mL of fermentation broth was centrifuged at 8000 rpm for 5 minutes, and the cells were harvested. The cells were washed and resuspended in PBS buffer (pH 7.4). Cells were disrupted by ultrasound (270 W, 4 s / 5 s, 20 min). The cell fragments were centrifuged at 12000 rpm for 3 minutes, and the supernatant was removed to obtain the crude enzyme solution.

[0054] Acetaldehyde dehydrogenase activity assay: Mix 30 μL of Tris-HCl (pH 9.2), 30 μL of coenzyme NAD, 20 μL of acetaldehyde, 10 μL of KCl, 3 μL of β-mercaptoethanol, and 197 μL of water. Preheat in a 35°C water bath for 10 minutes, then add 3 μL of crude enzyme solution. Measure the change in absorbance at 340 nm. Record readings every 1 minute for a total reaction time of 5 minutes. Acetaldehyde dehydrogenase activity is calculated using the following formula:

[0055] Acetaldehyde dehydrogenase activity = [(ΔA 340 ×μ) / (v×10 -3 )] / c, where the unit of acetaldehyde dehydrogenase activity is U / mL, where μ is the dilution factor of the enzyme, v is the amount of enzyme added, c is the protein concentration (mg / mL), and ΔA 340 It is the absorbance increase at 340 nm within 5 minutes.

[0056] The results are as follows Figure 14 As shown, the acetaldehyde dehydrogenase activity in the co-culture group (LPC01 + Yeast) was 36,703.3 U / mg, a significant increase of 11,490.6 U / mg compared to the LPC01 group. This indicates that the co-culture of lactic acid bacteria and yeast plays an important role in metabolic complementation and enzyme activity regulation. Yeast itself has high acetaldehyde dehydrogenase activity, especially during ethanol fermentation, where it converts acetaldehyde into acetic acid through its acetaldehyde dehydrogenase. Furthermore, related results indicate that the co-culture of yeast and lactic acid bacteria eliminates fishy odor by promoting dehydrogenase activity and enhancing the conversion of aldehydes to acids in the fermentation broth. Furthermore, the accumulation of organic acids improves the overall flavor attributes of the fermentation medium. Furthermore, the acidic metabolites of lactic acid bacteria further enhance the acetaldehyde dehydrogenase activity of yeast, resulting in superior performance in the co-culture system compared to lactic acid bacteria alone.

[0057] 4. Alcohol dehydrogenase activity assay: Take 1.5 mL of Tri-HCl buffer (pH 7.0, 0.05 mol / L), 0.5 mL of 40% ethanol solution and 1.0 mL of 4 mg / mL NAD +Solution was incubated at 37°C in a water bath for 20 minutes. Then, 0.1 mL of crude enzyme solution prepared as described above, preheated under the same conditions (in a 37°C water bath for 20 minutes), was added. The absorbance at 340 nm was read every 1 minute for 5 minutes. A 0.05 mol / L Tris-HCl buffer solution, pH 7.0, was used as a blank control. The alcohol dehydrogenase activity was calculated using the following formula:

[0058] Alcohol dehydrogenase activity = [(ΔA 340nm ×3.1) / (ΔX×0.1)] / c, where the unit of alcohol dehydrogenase activity is U / mg; where: ΔA 340 is the absorbance increase at 340 nm within 5 minutes; ΔX is the unit of absorbance increase

[0059] (0.001); 3.1 is the total reaction volume; 0.1 is the volume of the sample enzyme solution; c is the protein concentration (mg / mL).

[0060] The results are as follows Figure 15 As shown in the figure, in the LPC01+Yeas group, the enzyme activity of alcohol dehydrogenase reached 4148.0 U / mg, which was 1800.9 U / mg higher than that of the LPC01 group, indicating that the co-culture of lactic acid bacteria and yeast significantly promoted the catalytic activity of alcohol dehydrogenase. This result shows that the co-culture of lactic acid bacteria and yeast regulates the cofactor NAD through interaction. + The supply of NAD effectively enhances the catalytic activity of alcohol dehydrogenase. + As a key cofactor in the alcohol dehydrogenase reaction, it participates in the regulation of redox reactions during ethanol conversion. By jointly regulating metabolic pathways, lactic acid bacteria and yeast can complement each other, providing sufficient energy and cofactors to significantly enhance the enzymatic activity of alcohol dehydrogenase, thereby strengthening the overall activity of bacterial metabolic enzymes and improving the conversion efficiency of fatty aldehydes.

[0061] 5. Protease activity assay

[0062] Extraction of crude extracellular enzyme solution: Centrifuge 50 mL of fermentation broth at 10,000 rpm for 20 min at 4°C. Add ammonium sulfate to the supernatant to 80% saturation to precipitate the protease. Stir continuously to fully dissolve the protease. Let stand at 4°C for 12 h to allow the protease to fully precipitate. Collect the precipitate by centrifugation at 10,000 rpm for 20 min at 4°C. Redissolve the precipitate in 0.02 M PBS buffer (pH 7.0). Centrifuge again to collect the supernatant to obtain the crude extracellular enzyme solution.

[0063] Crude intracellular enzyme solution: Centrifuge 50 mL of fermentation broth at 10,000 rpm for 10 min at 4°C to collect the cells. Wash the cells twice with PBS buffer and collect them by centrifugation. Mix the cells with 5 mL of PBS buffer and disrupt them by ultrasonication in an ice-water bath (550 W, 6 s / 5 s, 6 min). Centrifuge the cell disruption solution at 10,000 rpm for 10 min at 4°C. Collect the supernatant to obtain the crude intracellular enzyme solution.

[0064] Protease activity assay: Place 1 mL of extracellular or intracellular crude enzyme solution in a 10 mL centrifuge tube and heat in a 40°C water bath for 2 minutes. Add 1 mL of preheated 2 wt% casein solution and maintain temperature for 10 minutes. Add 2 mL of 0.4 mol / L trichloroacetic acid to terminate the reaction and place in an ice bath for 10 minutes. Centrifuge at 4°C, 10,000 rpm for 10 minutes to collect the supernatant. Add 5 mL of 0.4 mol / L Na2CO3 and 1 mL of Folin-phenol reagent to 1 mL of supernatant and incubate in a 40°C water bath for 20 minutes. Measure the absorbance at 680 nm. Protease activity calculation formula:

[0065] Protease activity (U / g) = (A×V×4×n) / m×(1 / 10), where: A is the activity of the final sample dilution obtained from the standard curve, U / mL; V is the volume of the volumetric flask used for the solution sample, mL; 4 is the total volume of the reaction reagent, mL; n is the dilution factor of the sample; m is the mass of the sample, g; 1 / 10 is the reaction time of 10 min, calculated as 1 min.

[0066] The results are as follows Figure 16 and Figure 17 As shown, the intracellular and extracellular protease activities in the LPC01+Yeast group were 139.4 U / g and 387.1 U / g, respectively, representing increases of 6.1 U / g and 89.2 U / g, respectively, compared to the LPC01 group. This result further confirms that the synergistic effect between lactic acid bacteria and yeast plays a key positive feedback role in regulating protease activity. During co-culture, lactic acid bacteria produce organic acids (such as lactic acid and acetic acid), which lower the pH and promote increased protease synthesis in yeast. Conversely, yeast metabolites (such as alcohols and esters) may also stimulate protease synthesis in lactic acid bacteria. This metabolic interaction and synergistic effect leads to increased protease activity. Furthermore, resource competition within the co-culture environment further promotes the secretion of proteases by microorganisms to effectively degrade external proteins and optimize nutrient utilization, thereby increasing overall protease activity.

[0067] In summary, these results indicate that the co-culture of yeast and lactic acid bacteria significantly increases the activity of dehydrogenase, thereby promoting the degradation rate of fatty aldehydes under the co-culture conditions of the LPC01+Yeast group, and further promoting the efficient degradation of fishy aldehydes.

[0068] Specific Example 3: Effect of strain LPC01 on degradation of fishy smell in duck liver fermentation.

[0069] 1. Preparation of fermented duck liver: After removing the fascia from fresh duck liver, the liver was minced in a meat grinder to obtain duck liver paste. A yeast suspension was inoculated into the duck liver paste and fermented at 30°C for 1 h. The paste was then inoculated with a lactic acid bacteria suspension and fermented for 2 h. The yeast and lactic acid bacteria inoculation volume ratio was 1:1, and the total inoculation amount was 1 vt%, recorded as the LPC01+Yeast group.

[0070] Comparative Example 1: After removing the fascia from fresh duck liver, the liver was minced in a meat grinder to obtain duck liver paste; a lactic acid bacteria suspension was inoculated into the duck liver paste at an inoculum rate of 1vt%, and fermented at 30°C for 1h, which was recorded as the LPC01 group.

[0071] Comparative Example 2: Fresh duck liver was removed of its fascia and minced in a meat grinder to obtain duck liver paste, designated as the CK group.

[0072] 2. Determination of Volatile Compounds: 5 g of fermented duck liver was transferred to a 20 mL headspace vial. A 50 / 30 μm DVB / CAR / PDMS extraction tip was inserted into the vial to extract the volatile flavor compounds (50°C for 30 min). The extraction needle was then inserted into the inlet of a gas chromatograph (GC) at 250°C for desorption for 5 min. GC conditions included an inlet temperature of 250°C, splitless mode, He carrier gas at a flow rate of 0.8 mL / min, and a VLCOL column (60 m × 0.32 mm × 1.8 μm). The temperature program was as follows: start at 35°C and hold for 3 min; then increase the temperature at 3°C / min to 40°C, hold for 1 min, and then increase the temperature at 5°C / min to 210°C and hold for 11 min. Mass spectrometry (MS) conditions: ion source temperature 230°C, EI mode with 70 eV electron impact, Scan mode for qualitative and quantitative detection of volatile flavor compounds, with a detection range of m / z 45-600. Unknown volatile flavor compounds in duck liver protein were identified by comparing their mass spectra with known compounds in the NIST17 mass spectral database.

[0073] The results are as follows Figure 18 、 Figure 19 and Figure 20As shown in the data, a total of 25 volatile compounds were detected during the fermentation of duck liver, among which the CK group was mainly aldehyde compounds, including benzaldehyde (52.3%), hexanal (4.72%), eicosanal (4%), octanal (3.69%), and phenylacetaldehyde (1.41%). After the strain LPC01 was fermented alone, 11 compounds were detected, and the total concentration of aldehydes decreased by 42.14%, indicating that the strain LPC01 had a significant degradation effect on duck liver aldehyde compounds during the fermentation process, especially the removal of eicosanal, phenylacetaldehyde and hexanal, reflecting its obvious effect in inhibiting fishy compounds. Compared with the CK group and LPC01 group, the relative content of aldehyde compounds under co-culture conditions (LPC01+Yeast) decreased by 64.62% and 22.48%, respectively. This indicates that the mixed culture mode of yeast and lactic acid bacteria has a strong ability to metabolize and transform duck liver aldehydes, effectively reducing the concentration of aldehydes. It has important application prospects in improving the flavor properties of duck liver and reducing the perception of fishy smell. At the same time, it confirms the adaptability and application effect of the fatty aldehyde degradation law in the fermentation broth system in food matrices such as duck liver.

[0074] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. Application of a plant lactobacillus combination for degrading low molecular weight aldehydes in degrading the fishy smell of duck liver, characterized in that: The combination is composed of equal volumes of plant lactobacillus suspension and yeast suspension. The OD of the plant lactobacillus suspension is 600 The value is 0.5-2, and the OD of the yeast suspension is 600 The value is 0.5-2, and the plant lactobacillus is the LPC01 strain, which is classified and named as plant lactobacillus ( Lactobacillus plantarum ), was deposited in the General Microbiology Center of China Culture Collection Administration Committee on September 19, 2024, with the deposit number CGMCC No.32002. The yeast is Rhodotorula glutinosus ( Rhodotorula mucilaginosa ).

2. The use of a plant lactobacillus combination for degrading low molecular weight aldehydes according to claim 1 in degrading the fishy smell of duck liver, characterized in that: The yeast suspension is inoculated into the duck liver paste and fermented at 25-35°C for 0.5-1.5 hours, and then the Lactobacillus plantarum suspension is inoculated and fermented for 1.5-2.5 hours. The inoculation volume ratio of the yeast suspension to the Lactobacillus plantarum suspension is 1:1, and the total inoculation amount is 0.5-1.5vt%.

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