Lactobacillus sakei INM3103 producing 3-phenyl-2-propenoic acid and preparation and application thereof

Abstract

The application provides a 3-phenyl-2-propenoic acid producing Latilactobacillus sakei INM3103 preparation and application, the strain is Latilactobacillus sakei INM3103, which is preserved in the Guangdong Microbial Culture Collection Center on March 11, 2024, and the preservation number is GDMCC NO:64406.The advantage is that the concentration of cinnamic acid after whole grain sourdough fermentation can be effectively improved.

Description

Technical Field

[0001] This invention relates to the preparation and application of Lactobacillus 3-phenyl-2-acrylic acid-producing sake strain INM3103, which is suitable for enhancing cinnamic acid production during the fermentation stage of whole grain sourdough starter. Background Technology

[0002] 3-Phenylacetic acid, also known as cinnamic acid or β-phenylacrylic acid, is an organic compound with the chemical formula C9H8O2 and a molecular weight of 148.159. It is a white to pale yellow powder, an organic acid isolated from cinnamon bark or benzoin. It is produced in plants from the deamination and degradation of phenylalanine.

[0003] Cinnamic acid, with its anti-mold, antiseptic, and antibacterial properties, can be used for the preservation and sterilization of grains, vegetables, and fruits. In candied fruits, cinnamic acid improves taste and flavor, and it is particularly effective as a harmless and environmentally friendly preservative. Alternatives (such as sodium benzoate and potassium sorbate) can also be used in wine to enhance its color.

[0004] Lactic acid bacteria also have a deamination effect on amino acids. Ohshima (1987) reported that *L. plantarum* can deaminate serine to produce pyruvate, and Winters et al. (2000) reported that *L. paracasei* subsp. *paracasei* can degrade proline. Furthermore, the main function of lactic acid bacteria is to produce organic acids, which contribute to different flavors and preservative properties of products. Therefore, under specific conditions, lactic acid bacteria that produce specific organic acids can be screened out, thereby enhancing the unique flavor of the product.

[0005] *Lactobacillus sakei* (formerly known as *Lactobacillus sakei*) is a Gram-positive rod-shaped bacterium belonging to the genus *Lactobacillus*. As a typical example of facultative heterolactic fermentation, it can survive in harsh environments, exhibiting strong resistance to gastric acid and bile salts, allowing it to thrive and multiply well in the intestinal environment. Furthermore, *Lactobacillus sakei* can produce large amounts of organic acids (such as lactic acid, acetic acid, and fumaric acid), enzymes, and other beneficial substances, playing a crucial role in promoting intestinal health.

[0006] Dietary fiber promotes intestinal peristalsis and provides nutrients and a suitable growth environment for beneficial gut bacteria. Lactobacillus cereus, a type of bacteria found in sake, also requires dietary fiber to maintain normal growth. Whole wheat flour is rich in dietary fiber; therefore, fermenting whole wheat flour with Lactobacillus cereus not only increases the variety of organic acids and produces abundant flavor compounds, but also promotes its growth.

[0007] Currently, research on the production of cinnamic acid by lactic acid bacteria is relatively limited. Patent CN201310097036.1 discloses a method for synthesizing natural cinnamic acid, using cinnamaldehyde, a large amount isolated from cinnamon oil, as a raw material, and synthesizing natural cinnamic acid in a sodium dihydrogen phosphate buffer solution using hydrogen peroxide reagent and a catalyst. This method features short reaction steps, safe operation, high product purity, and suitability for large-scale production, making it suitable for industrial applications. Patent CN200910101842.5 discloses a method for preparing cinnamic acid in ionic liquids, using a sulfated ester-based ionic liquid as a catalyst and reaction solvent to catalyze the production of cinnamic acid from 1,3,3,3-tetrachlorophenylpropane.

[0008] Patent: A novel method for the catalytic oxidation of cinnamaldehyde to synthesize cinnamic acid - CN200610019615.4. This invention relates to a method for synthesizing sodium cinnamate from cinnamaldehyde under alkaline conditions using silver / copper as a catalyst and oxygen as the oxidant. The sodium cinnamate is then subjected to acid precipitation to generate crystalline cinnamic acid. This method is simple, has a recovery rate of over 95%, is inexpensive, and has good reusability.

[0009] The above patents all use chemical synthesis methods to generate cinnamic acid. Although it is a food additive permitted by national standards, chemically synthesized food additives are increasingly resisted by consumers. Therefore, it is clear that developing a safe and effective strain to increase cinnamic acid in whole grain products has a very broad application prospect. Although many studies have shown that there are effective methods to increase cinnamic acid content, there are still shortcomings in practical applications: (1) At present, there is still no method for producing cinnamic acid by lactic acid bacteria fermentation; (2) There is a lack of a safe strain that can be directly applied to increase cinnamic acid content during the whole grain fermentation stage. Summary of the Invention

[0010] This invention addresses the above-mentioned problems by providing a method for preparing and applying a strain of *Lactobacillus sacchariformis* INM3103 that produces 3-phenyl-2-acrylic acid. Without altering existing processes, this method utilizes *Lactobacillus sacchariformis* to ferment whole-grain sourdough starter, thereby enhancing the nutritional value of whole-grain products. This not only increases the cinnamic acid content in whole-grain products but also improves their flavor and quality. The fermentation process of whole-grain products produced using this preparation does not affect the overall process of the whole-grain products, thus improving both their taste and nutritional value.

[0011] The invention aims to achieve the following: a strain of *Latilactobacillus sakei* producing 3-phenyl-2-acrylic acid, strain INM3103, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 11, 2024, with accession number GDMCCNO:64406.

[0012] Furthermore, the colony characteristics of *Lactobacillus sacchariflorus* are as follows: After 48 hours of culture on solid MRS agar plates, the colonies are round, smooth, dense, milky white or milky yellow, 1.8-2.0 mm in size; the cells are rod-shaped, paired or in short chains, 0.5-0.9 × 0.6-1.5 μm in size, and do not form spores.

[0013] A method for preparing *Lactobacillus thuringiensis* that produces 3-phenyl-2-acrylic acid in sake is as follows: (1) Isolation of lactic acid bacteria: Lactic acid bacteria are isolated and screened from sauerkraut samples. The sauerkraut samples are processed into a paste and added to sterile water to dilute to a concentration of 10. 5 -10 6 Different concentrations of sauerkraut slurry were evenly spread on solid MRS medium and incubated at 37°C for 48 hours. White colonies and lactic acid bacteria with the correct morphology were selected from the solid MRS medium for screening.

[0014] (2) Screening of lactic acid bacteria: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated at a rate of 1%, with a final strain concentration of 1×10⁻⁶. 6 The cfu / mL inoculum was inoculated into the induction culture medium and incubated at 37℃ for 2 days to obtain the fermentation broth; the cinnamic acid content was then measured.

[0015] Furthermore, the formulation of the induction culture medium is as follows: weigh 4g yeast extract, 10g glucose, 1g phenylalanine, 0.04g manganese sulfate, and 1g Tween 80, dissolve them in 1000mL distilled water, and sterilize at 0.1MPa and 121℃ for 15min.

[0016] The application of a type of Lactobacillus that produces 3-phenyl-2-acrylic acid in sake can effectively increase the concentration of cinnamic acid in whole grain sourdough after fermentation.

[0017] Advantages of the present invention: (1) The Lactobacillus thuringiensis in sake described in the present invention can effectively increase the concentration of cinnamic acid after fermentation of whole grain sourdough;

[0018] (2) The sake described in this invention is widely covered with lactobacillus, which enhances the aroma and taste of whole grain products;

[0019] (3) The strain screening method of the present invention is universal and can be widely used to screen strains that can improve the cinnamic acid content in whole grain products;

[0020] (4) The Lactobacillus NS3504 for sake provided by this invention contains substances that can enhance the flavor of whole grains, and there is no off-flavor in the fermented whole grains. It can be mixed with other grain powders in the later stage, which preserves the nutrition and flavor of whole grains and has no safety risks. Attached Figure Description

[0021] Figure 1This is a liquid phase chromatogram for detecting cinnamic acid produced by NS3504.

[0022] Figure 2 This is a morphological observation of strain NS3504 on solid MRS agar plates.

[0023] Figure 3 This is an image of NS3504 strain observed under a 1000x microscope. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments:

[0025] Unless otherwise specified, the experimental methods used in the following implementation examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified; and the percentages mentioned are mass percentages unless otherwise specified.

[0026] Example 1, see attached document Figure 1-3 A method for preparing *Lactobacillus thuringiensis* that produces 3-phenyl-2-acrylic acid in sake.

[0027] 1. Raw material processing

[0028] Take the sauerkraut juice product and dilute it with sterile saline to a concentration of 10. -1 10 -2 10 -3 10 -4 10 -5 10 -6 To determine the concentration, prepare bacterial cell dilutions. Take 0.1 mL of each dilution and spread it evenly on MRS solid medium plates. Incubate at 37°C for 48 h. Select single colonies that are white, raised, smooth, and have neat edges. Streak the colonies on MRS solid medium for secondary purification. Incubate at 37°C for 48 h. Select single colonies for glycerol preservation and name them sequentially from NS3501 to NS3509.

[0029] 2. Preliminary screening of bacterial strains

[0030] The nine bacterial strains NS3501–NS3509 were inoculated into induction liquid medium at a 1% inoculum rate and cultured at 37°C for 24 hours. Then, they were inoculated into MRS medium at a 1% inoculum rate and cultured statically at 37°C for 24 hours until the final bacterial density reached 10⁻⁶. 6 cfu / mL.

[0031] After 24 hours of fermentation, samples were taken from each tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant obtained was used as the sample for physicochemical analysis. The results are shown in Table 1.

[0032] Induction culture medium: Weigh 4g yeast extract, 10g glucose, 1g phenylalanine, 0.04g manganese sulfate, and 1g Tween 80, dissolve in 1000mL distilled water, and sterilize at 0.1MPa and 121℃ for 15min.

[0033] Cinnamic acid detection: High performance liquid chromatography (HPLC) was used. Mobile phase: acetonitrile:water:acetic acid = 75 / 24 / 1; flow rate: 1 mL / min; detection wavelength: 275 nm; column temperature: room temperature; column: CAPCELL PAK ADME (4.6 × 250 mm); sample loading volume: 10 μL (sample and standard dissolved in 50% methanol); results are as follows. Figure 1 .

[0034] Table 1. Analysis of total acid and cinnamic acid produced during fermentation.

[0035]

[0036]

[0037] Strains with high total acid and cinnamic acid content, NS3502, NS3504, and NS3508, were selected for secondary screening.

[0038] 3. Secondary screening of strains

[0039] The initially screened lactic acid bacteria strains NS3502, NS3504, and NS3508 were reactivated and cultured at a bacterial concentration of 10. 6 The starter culture was prepared according to the national standard GB / T14612-2008 using cfu / mL, and then made into whole grain bread for sensory evaluation. The results are shown in Table 2.

[0040] Table 2 Sensory Analysis of Fermented Bread

[0041] NS3502 4.0 4.5 4.25 NS3504 5.0 4.8 4.9 NS3508 5.0 4.0 4.5

[0042] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.

[0043] As shown in Table 2, NS3504 had the best sensory evaluation results. As a target strain for secondary screening, the morphological observation of this strain on solid MRS plates is as follows: Figure 2 As shown, colonies are round, smooth, dense, milky white or milky yellow, 1.8-2.0 mm in size; cells are rod-shaped, paired or in short chains, 0.5-0.9 × 0.6-1.5 μm, and do not form spores. Results are as follows... Figure 3 As shown.

[0044] The NS3504 strain obtained through the above screening was identified by 16S rDNA sequencing. Comparative analysis showed that the 16S region sequence of the strain obtained in this invention had a 99.93% similarity to *Latilactobacillus sakei*, confirming that this strain is indeed *Latilactobacillus sakei* and can be used in the food fermentation industry. It was named *Latilactobacillus sakei* INM3103 and deposited at the Guangdong Provincial Microbial Culture Collection Center on March 18, 2024, with accession number GDMCC No. 64406. Therefore, in this application, INM3103 and NS3504 refer to the same strain. The gene sequence of the strain INM3103 of this invention is as follows:

[0045]

[0046] Example 2, a strain of Lactobacillus sacchariformis INM3103

[0047] Genetic stability assay of Lactobacillus saccharidus INM3103 in sake

[0048] The *Lactobacillus sacchariformis* INM3103 from Example 1 was passaged 10 times on induction liquid medium. The samples from the 1st, 5th, and 10th generations were centrifuged at 10,000 rpm for 10 minutes, and the supernatant was used as the sample. Physicochemical indicators were tested, and the methods for determining total acid content and cinnamic acid content were the same as in Example 1. Sensory evaluation was also performed to determine its passage stability; specific data are shown in Table 3.

[0049] Table 3. Results of the passaging stability test

[0050] INM3103 1st generation 45.56±0.22 31.23±0.18 5.0 INM3103 5th generation 44.41±0.18 32.20±0.29 5.0 INM3103 10th Generation 46.28±0.51 33.41±0.31 5.0

[0051] Table 3 shows that the fermentation performance of Lactobacillus spp. INM3103 strains from different generations of sake was normal, with the total acid and cinnamic acid content varying within 10%, indicating good genetic stability and meeting the requirements for production and use.

[0052] Example 3: A strain of Lactobacillus sacchariflorus INM3103

[0053] Using Lactobacillus spp. INM3103 sourdough starter in sake to make bread

[0054] According to national standards, Lactobacillus thuringiensis INM3103 was inoculated into the fermentation sourdough starter in sake.

[0055] GB / T14612-2008 method for preparing whole grain sourdough starter and whole grain bread, with inoculation concentration of fermentation bacteria at 10... 4 -10 8 At CFU / mL, whole grain bread was prepared separately, with NS3509 as the control strain. The method for detecting the total acid and cinnamic acid content in the sourdough starter was the same as in Example 1, and the results are shown in Table 4.

[0056] Table 4. Total acid content and cinnamic acid content in whole grain sourdough starter

[0057]

[0058]

[0059] Table 5 Sensory evaluation results of whole grain bread

[0060] Control group 1 <![CDATA[1.0×10 4 ]]> 6.0 6.2 7.5 19.7 Experimental group 1 <![CDATA[1.0×10 4 ]]> 6.5 7.4 8.2 22.1 Control group 2 <![CDATA[1.0×10 5 ]]> 7.4 7.8 9.3 24.5 Experimental group 2 <![CDATA[1.0×10 5 ]]> 6.9 8.4 8.3 23.6 Control group 3 <![CDATA[1.0×10 6 ]]> 7.2 8.0 7.5 22.7 Experimental group 3 <![CDATA[1.0×10 6 ]]> 9.1 9.0 9.2 27.3 Control group 4 <![CDATA[1.0×10 7 ]]> 5.5 6.4 8.0 19.9 Experimental group 4 <![CDATA[1.0×10 7 ]]> 9.5 9.7 9.6 28.8 Control group 5 <![CDATA[1.0×10 8 ]]> 6.3 7.4 8.6 22.3 Experimental group 5 <![CDATA[1.0×10 8 ]]> 8.7 7.9 8.3 24.9

[0061] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores.

[0062] As shown in Tables 4 and 5, the use of *Lactobacillus cereus* INM3103 in whole-grain bread significantly increased the total acid and cinnamic acid content of the sourdough starter, with higher inoculum sizes resulting in higher levels of both cinnamic acid and total acid. However, excessively high or low inoculum concentrations of *Lactobacillus cereus* INM3103 negatively impacted the bread's sourness, sweetness, and texture. The optimal inoculum concentration was 1.0 × 10⁻⁶. 7 CFU / mL, this concentration produces enough total acidity while also enhancing the flavor and texture of whole-grain bread.

[0063] Example 4: A strain of Lactobacillus sacchariflorus INM3103

[0064] Using Lactobacillus thuringiensis INM3103 sourdough starter from sake to make baguettes;

[0065] The final concentration of Lactobacillus widespread var. chinensis INM3103 in sake was 10. 7 The whole grain sourdough inoculum concentration was prepared using a dose of CFU / mL. Another group was inoculated with NS3509 as a control group. Bagels were then prepared according to the national standard GB / T14612-2008.

[0066] After fermentation, the total acid content and cinnamic acid of the whole grain baguette were tested according to the method in Experiment Example 1. At the same time, a professional evaluator was asked to conduct a sensory evaluation of the baguette flavor. The results are recorded in Table 6.

[0067] Table 6. Results of Whole Grain Sourdough Detection and Sensory Evaluation of Baguettes

[0068]

[0069] Note: Each sensory evaluation item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all items.

[0070] As shown in Table 6, the results indicate that whole grains were inoculated for 10 days... 7 Using CFU / mL Lactobacillus globosum INM3103 in sake to make sourdough starter can significantly increase the total acid content and cinnamic acid content of fermented sourdough starter; in addition, it can improve the sensory evaluation of baguette, increase baguette aroma, enhance baguette flavor and texture, and make baguette fragrant and sweet and sour.

[0071] Example 5: A strain of Lactobacillus sacchariflorus INM3103

[0072] Using Lactobacillus thuringiensis INM3103 sourdough starter from sake to make croissants;

[0073] The final concentration of Lactobacillus widespread var. chinensis INM3103 in sake was 10. 7The whole grain noodle starter concentration was prepared using a dose of CFU / mL. Another group was inoculated with NS3509 as a control group. Croissants were then prepared according to the national standard GB / T14612-2008.

[0074] After fermentation, the total acid content and cinnamic acid content of the whole grain sourdough starter were tested according to the method in Experiment Example 1. At the same time, a professional evaluator was asked to conduct a sensory evaluation of the croissant flavor. The results are recorded in Table 7.

[0075] Table 7. Results of Whole Grain Pasta Starter Testing and Sensory Evaluation of Croissants

[0076]

[0077] Note: Each sensory evaluation item is scored out of 10 points, with a scoring interval of 0.1. The overall score is the sum of all items.

[0078] As shown in Table 7, inoculating whole grains with 10 7 Using CFU / mL Lactobacillus thuringiensis INM3103 in sake to make sourdough starter can significantly increase the total acid content and cinnamic acid content of the fermented starter. In addition, it can improve the sensory evaluation of croissants, increase their aroma, enhance their flavor and texture, and make them fragrant, sweet and sour.

[0079] Example 6: A strain of Lactobacillus sacchariformis INM3103;

[0080] Bagels made with Lactobacillus thuringiensis INM3103 starter from sake;

[0081] The final concentration of Lactobacillus widespread var. chinensis INM3103 in sake was 10. 7 The whole grain sourdough inoculum concentration was prepared using a dose of CFU / mL. Another group was inoculated with NS3509 as a control group. Bagels were then prepared according to the national standard GB / T14612-2008.

[0082] After fermentation, the total acid content and cinnamic acid content of the whole grain sourdough starter were tested according to the method in Experiment Example 1. At the same time, a professional evaluator was asked to conduct a sensory evaluation of the croissant flavor. The results are recorded in Table 8.

[0083] Table 8 Sensory Evaluation Results of Whole Grain Sourdough and Bagels

[0084]

[0085] Note: Each sensory evaluation item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all items.

[0086] As shown in Table 8, inoculating whole grains with 10 7Using CFU / mL Lactobacillus globosum INM3103 in sake to make sourdough starter can significantly increase the total acid content and cinnamic acid content of the fermented starter. In addition, it can improve the sensory evaluation of bagels, increase bagel aroma, enhance bagel flavor and taste, and make bagels fragrant, sweet and sour and delicious.

[0087] Based on the results of the above embodiments, the Lactobacillus sakei INM3103 provided by the present invention, when applied to the fermentation of whole grain sourdough starter, can significantly increase the total acid content and cinnamic acid content of related whole grain products without changing the existing process. At the same time, it can also improve the aroma and taste of whole grain products, and significantly improve the quality of whole grain products.

[0088] Although the present invention has been described with reference to preferred embodiments, those skilled in the art will understand that it is not limited to the description of the above embodiments, and various changes in form and detail may be made within the scope of the claims.

[0089]

[0090]

Claims

1. A strain of *Lactobacillus thuringiensis* that produces 3-phenyl-2-acrylic acid sake, characterized in that: This strain is *Lactobacillus sacchari* (Sake Bacterium sacchari). Latilactobacillus sakei INM3103 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on March 11, 2024, with accession number GDMCC NO:64406.

2. The *Lactobacillus thuringiensis* strain producing 3-phenyl-2-acrylic acid sake according to claim 1, characterized in that: The colony characteristics of the *Lactobacillus sacchariformis* are as follows: After 48 hours of culture on solid MRS agar plates, the colonies are round, smooth, dense, milky white or milky yellow, 1.8-2.0 mm in size; the cells are rod-shaped, paired or in short chains, 0.5-0.9 × 0.6-1.5 µm in size, and do not form spores.

3. The application of *Lactobacillus thuringiensis* producing 3-phenyl-2-acrylic acid as described in claim 1, characterized in that: It can effectively increase the concentration of cinnamic acid after fermentation of whole grain sourdough.

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