A Lactobacillus plantarum strain producing fragrance and its application

Abstract

The present invention discloses a fragrance-producing Lactobacillus plantarum strain and its application. The fragrance-producing Lactobacillus plantarum strain is classified and named as Lactobacillus plantarum RP26, and its scientific name is Lactobacillus plantarum RP26 has been deposited with the China Center for Type Culture Collection under the CCTCC No. M 2019298, dated April 26, 2019. Lactobacillus plantarum RP26 is an excellent microorganism specifically designed for deodorizing tuna cooking liquid. It effectively reduces the unpleasant flavor of tuna cooking liquid and produces a unique fermented aroma, which is of great significance for the development of high-value-added tuna cooking liquid products.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to an aroma-producing Lactobacillus plantarum strain and application thereof. Background Art

[0002] Tuna, also known as bluefin tuna or tuna, is a large, pelagic, and important commercial edible fish. Known as the "gold of the ocean," it is one of the three most nutritious fish recommended by the World Nutrition Organization. It contains eight essential amino acids for the human body, as well as numerous nutrients and functional substances such as DHA and EPA, making it low in fat and nutritious. Tuna can be processed into sashimi, canned fish, and fish floss. During the processing of 1,000 kg of tuna fillets and canned tuna, approximately 300-500 kg of tuna cooking liquid is produced as a byproduct. This cooking liquid is rich in essential and easily absorbed amino acids, soluble proteins, nucleotides, unsaturated fatty acids, and other nutrients. The cooking liquid has a delicious flavor, with its distinctive seafood flavor, making it a high-quality raw material for the production of condiments, protein supplements, and other foods. However, its strong fishy odor significantly affects the taste of further processed products, limiting the high-value development and utilization of high-quality tuna cooking liquid. At present, tuna cooking liquid has not been well developed and utilized in my country. Most of it is discharged as wastewater without treatment, which not only greatly wastes protein resources, but also increases wastewater treatment costs and poses an environmental pollution risk.

[0003] Currently, the main methods for removing fishy smells from seafood include physical, chemical, and biological methods. Physical deodorization generally involves adding a certain amount of adsorbent to the raw materials, which absorbs odorous substances and achieves the desired effect. However, this method suffers from incomplete deodorization and is difficult to commercialize. Chemical deodorization utilizes compounds that contribute to unpleasant flavors in aquatic products through reactions with chemicals, but it also has the disadvantage of easily producing odors and hindering industrial application. Biological deodorization does not involve the addition of other additives or chemicals, and is harmless to the human body. Microbial fermentation not only effectively reduces unpleasant flavors in fermented products but also imbues them with unique, pleasant flavors, thereby enhancing the overall flavor value of the food. Gu Saiqi et al. used yeast to deodorize kelp, achieving a removal rate of up to 39.75% and achieving excellent results. Zhang Haiyan et al. used yeast extract to deodorize sea bass fillets, resulting in a fishy-free fish fillet with no impact on the color, texture, or freshness of the fillets. Yeast imparts a unique flavor and aroma to the fermented broth by increasing the content of aromatic compounds.

[0004] Different aquatic products contain different odorous substances, and the deodorizing microorganisms used are also different. Chinese patent application "A Method for Deodorizing Kelp Using Composite Microorganisms (ZL201710981772.1) discloses a kelp deodorizing microbial strain and its deodorizing method; Chinese patent application "A Fermented Seaweed Cake (ZL201810898673.1) discloses a Gracilaria microbial deodorizing strain and its deodorizing method; and Chinese patent application "A Method for Biological Deodorization of Shellfish Meat Using Ozone Combined with Oxygen-Isolated Microbial Fermentation" discloses a scallop microbial deodorizing agent and its deodorizing method. However, these strains have been ineffective in removing the odor from tuna cooking liquid. Summary of the Invention

[0005] The purpose of the present invention is to provide an aroma-producing Lactobacillus plantarum strain and application thereof.

[0006] In order to achieve the above object, the present invention adopts the following technical solutions:

[0007] The present invention provides a fragrance-producing Lactobacillus plantarum strain, which is classified and named Lactobacillus plantarum RP26, with a scientific name of Lactobacillus plantarum RP26, and has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCCNO: M 2019298 and a deposit date of April 26, 2019.

[0008] The flavor compounds in tuna cooking liquid are primarily fishy-smelling decanal, hexanal, and 1-octen-3-ol; greasy 2-nonanone; and sour-smelling heptanal, trimethylamine, and dimethyl trisulfide. Currently, there is a lack of effective microorganisms for removing the fishy smell from tuna.

[0009] The aroma-producing Lactobacillus plantarum RP26 provided by the present invention can be used to remove the fishy smell of tuna cooking liquid. This strain effectively removes 2-nonanone, trimethylamine, and dimethyl trisulfide from the tuna cooking liquid. The characteristic flavor substances produced are 2-butyl-2-octenal and 2-dodecenal, which have a grassy aroma, and 2-undecanone, which has a fruity aroma. The fermented liquid has no noticeable fishy smell and exhibits a unique flavor of floral, fruity, and grassy notes, as well as tuna.

[0010] The excellent tuna cooking liquid deodorizing microorganism Lactobacillus plantarum RP26 screened by the present invention can degrade the fishy substances in the tuna cooking liquid, reduce the unpleasant flavor of the tuna cooking liquid, and produce a unique fermentation aroma, which is of great significance for the research and development of high-value-added tuna cooking liquid processed products. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 This is the colony morphology of strain RP26.

[0012] Figure 2This is the microscopic morphology of strain RP26.

[0013] Figure 3 This is the sequencing result of 16S rDNA detection of strain RP26.

[0014] Figure 4 This is the phylogenetic tree of 16S rDNA sequences of strain RP26 and related species.

[0015] Figure 5 Flavor wheel for fermented tuna cooking liquid.

[0016] Figure 6 Average variable importance projection (VIP) values; red bars indicate VIP values >1, and green bars indicate VIP values <1; (A) VIP-CK is the VIP value of the tuna cooking liquid group (CK treatment), and (B) VIP-R corresponds to the VIP value of the lactic acid bacteria single-strain fermentation group (R treatment).

[0017] Figure 7 This is a correlation analysis of characteristic flavor substances of lactic acid bacteria. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0019] Example 1 Isolation, screening, identification and preservation of strains

[0020] 1. Isolation and purification of strains

[0021] Lactic acid bacteria were isolated from malic acid fermentation mash. Gradual dilutions were applied to plates containing lactic acid bacteria isolation and purification medium. Multiple streaking and microscopic examinations were performed to obtain relatively pure strains. The strains were isolated and purified using the streak plate method. Gram staining, catalase reaction, indole test, and sugar fermentation tests were performed to identify strains with typical lactic acid bacterial colony characteristics. These strains were designated RP21, RP22, RP23, RP24, RP25, RP26, RP27, and RP28. These strains were inoculated onto MRS slants, cultured at 28°C for 3 days, and stored in a refrigerator at 4°C for future use.

[0022] 2. Screening of strains

[0023] The isolated strains were added to rice saccharification liquid culture medium at an inoculum rate of 2%, and fermented at 20°C for 5 days. The fermentation flavor was used as an indicator to screen the aromatic lactic acid-producing strains.

[0024] Fermented samples were categorized into five levels based on their flavor characteristics: Level 1, representing a strong fermentation aroma, was labeled "+++++"; Level 2, representing a relatively strong fermentation aroma, was labeled "++++"; Level 3, representing a moderate fermentation aroma, was labeled "+++"; Level 4, representing a relatively weak fermentation aroma, was labeled "++"; and Level 5, representing the absence of a fermentation aroma, was labeled "-". Twenty trained professionals independently evaluated the samples, and the average values were used for statistical analysis.

[0025] Table 1 shows the results of fermentation flavor tests on eight strains in rice saccharification broth. Lactobacillus RP26 exhibited a strong fermentation aroma, rated "+++++." Lactobacillus RP21 and RP25 exhibited fermentation aroma, rated "++++" and "+++." Lactobacillus RP22, RP23, and RP24 exhibited a weaker fermentation aroma, while RP27 and RP28 failed to produce a fermentation aroma and were rated "-." These results indicate that RP26 was the superior lactic acid bacterium with a unique fermentation flavor.

[0026] Table 1 Evaluation of fermentation flavor of 12 yeast strains

[0027]

[0028] 3. Identification of strains

[0029] The strain RP26 was inoculated on an MRS plate and cultured at 30°C for 2 days. The colony morphology was observed to be milky white, round, with a smooth, convex, moist surface and neat edges. Figure 1 As shown. Figure 2 The cell morphology observed under the microscope was: rod-shaped, 0.5μm×1.0~1.67μm in size, arranged singly or in short chains, non-spore-forming, and Gram-positive.

[0030] The 16S rDNA of strain RP26 was detected and the sequencing results were as follows Figure 3 shown.

[0031] The MEGA4.1 software was used to display the 16S rDNA phylogenetic tree of strain RP26 and related species using the neighbor joining method, and the similarity was calculated 1000 times. Figure 4 Only the nodes in the developmental tree show the Bootstrap values greater than 50%, and the superscript "T" indicates the type strain (L., Lactobacillus B., Bifidobacterium).

[0032] Phylogenetic analysis showed that strain RP26 and the model strains of L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, L. paraplantarum, and L. pentosus were clustered in the same phylogenetic branch, and the sequence homology was greater than 99.4%.

[0033] Based on the gene sequencing results of the strain and combined with the physiological and biochemical characteristics of the strain, strain RP26 was identified as Lactobacillus plantarum RP26, which has been deposited in the China Center for Type Culture Collection with the deposit number CCTCC NO: M 2019298 and the deposit date is April 26, 2019.

[0034] Example 2 Application of Lactobacillus plantarum RP26

[0035] This example investigates the effect of Lactobacillus plantarum RP26 on removing the fishy smell of tuna cooking liquid.

[0036] 1. Breeding of superior strains for deodorizing tuna cooking liquid

[0037] 1 Materials and Methods

[0038] 1.1 Materials

[0039] 1.1.1 Strains

[0040] Lactic acid bacteria: Lactobacillus paracasei RYPC301 (CICC 22709), Lactobacillus paracasei subsp. paracasei RP38 (disclosed in Chinese patent application CN110656061A) and Lactobacillus plantarum RP26.

[0041] 1.1.2 Tuna Cooking Liquid

[0042] Tuna cooking liquid: Provided by Fujian Haihui Biotechnology Industrial Co., Ltd., filtered, aliquoted, and stored at -20°C until use. Cooking liquid pH 6.67; soluble solids 3.75%; total acid 0.238g / 100g, calculated as lactic acid (0.090); amino acid nitrogen 0.07g / kg; reducing sugar 0.032g / 100g; total sugar 0.089g / 100g; protein 2.84g / 100g; crude polysaccharides 0.0184g / 100g; hydroxyproline 0.236g / 100g.

[0043] 1.2 Test methods

[0044] 1.2.1 Preparation of culture medium

[0045] MRS liquid culture medium: used for lactic acid bacteria culture; sterilize at 121℃ for 30min, cool and set aside.

[0046] 1.2.2 Seed solution preparation

[0047] After the bacteria were activated, they were inoculated into appropriate culture medium and cultured until the bacterial count reached 10 8 cfu / mL, then centrifuge at 5500r / min and 4℃ for 10min, take the bacterial mud and add an equal volume of sterile water for standby use.

[0048] 1.2.3 Fermentation process

[0049] Take 100mL of cooking liquid and place it in a 250mL Erlenmeyer flask, add 2% (w / w) white sugar and 3g / L malic acid, sterilize in a 100℃ water bath for 10min and cool. 7 CFU / mL were inoculated into the strains selected in 1.1.1, and fermented at 30℃ for 72h. The fermentation broth was collected and centrifuged for later use. Each treatment was repeated 3 times.

[0050] 1.2.4 Sensory evaluation of fermentation flavor of different strains

[0051] The fermentation liquid obtained in 1.2.3 was used as the object, the tuna cooking liquid without added bacteria was used as the control (CK group), and the sensory evaluation was used as the measurement index to screen the yeast strain suitable for removing the fishy smell of tuna cooking liquid.

[0052] 1.2.5 Evaluation of RP26 deodorization effect

[0053] Take 100mL of cooking liquid and place it in a 250mL Erlenmeyer flask, add 20g / L white sugar and 3g / L malic acid, sterilize in a 100℃ water bath for 10min, and cool. 8 cfu / mL were inoculated into RP26 and fermented at 30 ± 1°C for 72 h. The fermentation broth was centrifuged at 8000 rpm for 5 min, and the supernatant was collected. The composition and content of characteristic volatile flavor compounds of each treatment were analyzed using HS-SPME-GC-MS, and flavor sensory evaluation was performed. Each treatment was replicated three times.

[0054] 1.2.6 Sensory evaluation method

[0055] Thirteen trained professionals conducted a profile description of tuna cooking liquid based on the "GB / T 10220 General Principles of Sensory Analysis Methodology". They selected fishy smell, sour smell, greasy smell, green aroma, floral aroma, fruity aroma, mellow aroma, and lactic acid fermentation aroma as flavor sensory descriptors. The evaluation team used a scale of "0-5" to quantitatively describe the flavor intensity and drew sensory profiles to compare the deodorization effects after fermentation with different bacteria.

[0056] 1.2.7 Data Processing

[0057] SPSS 20.0 software was used to perform variance analysis on the data.

[0058] 2 Results and Analysis

[0059] 2.1 Screening of suitable strains for deodorization of tuna cooking liquid

[0060] The flavor intensity was quantitatively described on a scale of 0-5. The flavor intensity was scored for the tuna cooking liquor after fermentation for 72 h using a single strain. Figure 5 The sour and greasy flavors of M6 (RP26 Lactobacillus plantarum) decreased, but M3 (RYPC301 Lactobacillus paracasei) and M5 (RP38 Lactobacillus paracasei subsp. paracasei) showed no significant changes. Considering the three aspects of fishy smell removal, aroma production, and stability, M6 (RP26 Lactobacillus plantarum) is the optimal strain for removing fishy smell from tuna cooking liquid fermentation.

[0061] 2.2 Analysis of the consumption and production of characteristic flavor substances by Lactobacillus plantarum RP26

[0062] Based on the threshold values reported in the prior art, the OAV values of the flavor components of tuna fermentation broth were calculated, and the results are shown in Table 2. The contents of the main volatile flavor substances in tuna cooking liquid and RP26 fermentation broth are shown in Table 1, and the VIP value analysis results are shown in Figure 6 Based on the VIP and OAV analysis (OAV value ≥ 1 and VIP value > 1), and the description of flavor compounds in Table 2, the flavor compounds in the CK group (CK treatment) of tuna cooking liquid were primarily fishy-smelling decanal (6.35), hexanal (3.62), and 1-octen-3-ol (3.18); greasy-smelling 2-nonanone (26.10); and sour-smelling heptanal (2.30), trimethylamine (6.62), and dimethyl trisulfide (3.41). These are the primary components of the fishy odor of tuna cooking liquid. In addition, the CK group also contained some flavor compounds, such as 2-ethyl-furan (11.03) and 2-pentyl-furan (14.91), which have a fishy flavor. The main flavor substances of the lactic acid bacteria fermentation group are 2-dodecenal (2.01) and 2-butyl-2-octenal (1.52) with grassy aroma; 2-undecanone (5.32) with fruity aroma; and 2-ethyl-furan (21.47) and 2-pentyl-furan (15.61) with fishy aroma.

[0063] Table 1 Changes in flavor substances before and after RP26 fermentation

[0064]

[0065]

[0066]

[0067]

[0068]

[0069] Table 2 Volatile flavor substances with OAV>1

[0070]

[0071] 2.2 Contribution of lactic acid bacteria to the evolution of characteristic flavor compounds in tuna cooking liquid

[0072] The characteristic flavor substances fermented by Lactobacillus plantarum RP26 were analyzed by Sperman correlation analysis (P<0.05 and correlation |r|>0.5). Figure 7 Based on the Opls-da analysis, Spearman correlation analysis, OAV analysis, and the flavor descriptions of related substances in Table 2, it was determined that the flavor substances with VIP values > 1.0, correlation |r| > 0.5, and OAV > 1 were the characteristic flavor substances of tuna cooking liquid microbial fermentation. The results showed that the flavor characteristic substances consumed by Lactobacillus plantarum RP26 were 2-nonanone with an oily flavor, trimethylamine and dimethyl trisulfide with a sour and smelly smell; the characteristic flavor substances produced were 2-butyl-2-octenal and 2-dodecenal with a grassy aroma, and 2-undecanone with a fruity aroma.

Claims

1. A fragrance-producing Lactobacillus plantarum strain, characterized by: Its classification name is Lactobacillus plantarum RP26, and its scientific name is Lactobacillus plantarum RP26 has been deposited in the China Center for Type Culture Collection with the deposit number CCTCCNO: M 2019298 and the deposit date is April 26, 2019.

2. Application of the aroma-producing plant lactobacillus strain as claimed in claim 1 in removing fishy smell from tuna cooking liquid.

Images