Preparation and application of a strain of paracasei inm3110 for improving the flavor of whole grain sourdough
By screening and applying Lactobacillus paracasei INM3110 in the fermentation of whole grain sourdough, the problem of insufficient eugenol and butyl hexanoate content in existing technologies has been solved, achieving a safe and effective improvement in flavor and texture.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG INM FOOD CO LTD
- Filing Date
- 2024-05-20
- Publication Date
- 2026-07-31
AI Technical Summary
There is a lack of a safe and effective lactic acid bacteria strain that can increase the content of eugenol and butyl hexanoate during the fermentation stage of sourdough, and the relevant microorganisms are not on the list of edible fungi, so the chemical synthesis method is resisted by consumers.
A strain of Lactobacillus paracasei INM3110 was provided, which was screened from kimchi and applied to whole grain sourdough during fermentation. The fermentation time was 3 days, the temperature was 37℃, and the inoculum size was 104-108 cfu/mL, which increased the content of eugenol and butyl hexanoate.
It significantly increases the content of eugenol and butyl hexanoate in whole grain products, improves the flavor and texture of dough, and meets food safety standards without changing the existing process.
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Figure CN118360213B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the preparation and application of Lactobacillus paracasei INM3110, a strain suitable for producing eugenol and butyl hexanoate during the fermentation stage of sourdough to improve the flavor of whole grain sourdough. Background Technology
[0002] Syringate acid (chemical name: 3,5-dimethoxy-4-hydroxybenzoic acid, molecular formula: C9H10O5, molecular weight: 198.18) has a strong aroma and is therefore often used as a fragrance. Syringate acid has many important applications. First, it is an important intermediate compound used to prepare other organic compounds, such as methyl syringate and ethyl syringate. Second, syringate acid is an effective antioxidant component of traditional Chinese medicine and a new type of nutritional food. It has potential activities such as anti-angiogenesis, anti-glycation, anti-hyperglycemia, neuroprotection, and memory enhancement.
[0003] Butyl hexanoate, with the molecular formula C10H20O2 and a molecular weight of 172.27, has an elegant aroma of pineapple and apple, with hints of green and waxy notes. Its flavor profile includes sweet jackfruit and a slightly fermented fruity taste.
[0004] *Lactobacillus paracasei* is a pleomorphic bacillus, varying in length from short to long rod-shaped, but generally less than 1.5 μm wide. The bacteria are square-shaped with both ends flat, and are arranged in short or long chains. Spherical bacteria are sometimes also observed. It is Gram-positive, non-motile, and does not produce spores. *Lactobacillus paracasei* belongs to the *Lactobacillus* group within the genus *Lactobacillus* and is widely found in fermented foods such as cheese and pickles. It possesses the probiotic functions of *Lactobacillus casei*, including regulating the gut and enhancing immunity, and its bacteriocins have excellent antibacterial properties. Therefore, it plays an important role in the food and healthcare industries.
[0005] Currently, there is limited research on the production of syringic acid and ethyl hexanoate by *Lactobacillus paracasei* fermentation. Patent: Preparation of Syringic Acid - CN202011430480.7. This invention relates to a method of obtaining syringic acid by dissolving p-hydroxybenzoic acid in glacial acetic acid and an aqueous solution, using ferric chloride as a catalyst, and through a system of temperature control, cooling, and crystallization. However, it does not involve the production of syringic acid by lactic acid bacteria fermentation. Patent: An Extraction Method for Syringic Acid - CN201110045089.7. This invention relates to the extraction and recovery of syringic acid by fully impregnating oats under supercritical conditions and adjusting temperature, pressure, and time. However, it does not involve the lactic acid bacteria fermentation process.
[0006] Patent: A pickled pepper flavoring and its preparation method - CN202211360902.7. This patent relates to a pickled pepper flavoring formula composed of flavorings such as butyl hexanoate and ethyl acetate, but does not involve lactic acid bacteria fermentation, thus its application direction differs from this patent. Patent: A high-temperature koji enhanced with compound microbial agents and its application in soy sauce-flavored baijiu - CN202210532280.5. This invention relates to a microbial agent consisting of brewing yeast, acid-resistant lactobacillus, Bacillus licheniformis, and Bacillus subtilis fermenting baijiu to produce butyl hexanoate. However, this patent does not involve lactic acid bacteria producing butyl hexanoate, and apart from brewing yeast, the other strains are not listed in the "List of Edible Fungi," therefore, it cannot be effectively applied in actual production and processing.
[0007] The above patents all utilize chemical synthesis and natural extraction to produce eugenol, but the addition of chemical flavorings to enhance product flavor is increasingly met with consumer resistance. Furthermore, although butyl hexanoate obtained through microbial preparation methods is available, it is not on the nationally regulated list of microbial strains for application. Therefore, developing a safe and effective strain to enhance the nutrition and flavor of sourdough has broad application prospects.
[0008] Although studies have shown that methods can effectively enhance syringic acid and butyl hexanoate, there are still shortcomings in practical applications: (1) The microorganisms involved in the production of butyl hexanoate by microbial methods are not on the list of edible fungi and cannot be used in food. (2) In industrial applications, there is still a lack of a standard lactic acid bacteria strain that can be used to enhance syringic acid and butyl hexanoate in the fermentation stage of sourdough. Summary of the Invention
[0009] To address the above problems, this invention provides a method for preparing and applying Lactobacillus paracasei INM3110, a strain that improves the flavor of whole-grain sourdough. Without altering existing processes, this method enhances the content of eugenol and butyl hexanoate in noodle products by fermenting sourdough starter with Lactobacillus paracasei.
[0010] The invention aims to achieve the following: a strain of *Lactobacillus paracasei* that improves the flavor of whole-grain sourdough.
[0011] The strain is Lacticaseibacillus paracasei INM3110, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCCNO:64515.
[0012] Furthermore, the colony characteristics of *Lactobacillus paracasei* are as follows: after culturing on MRS solid medium for 48 hours, the colonies are raised, round, smooth, dense, and white.
[0013] A method for preparing *Lactobacillus paracheirica* to improve the flavor of whole-grain sourdough is as follows:
[0014] (1) Isolation: Lactic acid bacteria were isolated and screened from the kimchi. The kimchi was processed into a paste and added to sterile water to dilute the concentration to 10. 5 -10 6 Different concentrations of kimchi were evenly spread on MRS solid medium containing 0.015% bromocresol purple and incubated at 37°C for 48 hours. White colonies and lactic acid bacteria with the correct morphology were selected from the MRS solid medium for screening.
[0015] (2) Screening: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated at a 1% inoculum, with a final strain concentration of 1×10⁻⁶. 6 The concentration of cfu / mL was inoculated into the induction solution, and the mixture was incubated at 37℃ for 2 days to obtain the fermentation broth. The contents of syringic acid and butyl hexanoate were measured and selected.
[0016] Furthermore, the induction solution was mulberry juice, sterilized at 60°C for 30 minutes.
[0017] The application of a strain of *Lactobacillus paracheirica* that improves the flavor of whole-grain sourdough: Based on the whole-grain product manufacturing process, the inoculum quantity in the fermented whole-grain liquid was 10... 4 -10 8 Lactobacillus paracasei (cfu / mL) was fermented for 3 days at a temperature of 37°C.
[0018] Application of a strain of *Lactobacillus paracasei* that effectively enhances the production of higher concentrations of eugenol and butyl hexanoate after fermentation of sourdough.
[0019] Advantages of the present invention: (1) The screening method is universal and can be widely used to screen strains that reduce the content of eugenol and butyl hexanoate in whole grain products.
[0020] (2) It contains substances that can enhance the flavor of sourdough and there will be no off-flavor in the fermented dough. It can be mixed with other flours in the later stage, which preserves the nutrition and flavor of the flour and has no safety risks.
[0021] (3) The Lactobacillus paracasei can effectively enhance the production of higher concentrations of eugenol and butyl hexanoate after fermentation of sourdough and improve the aroma and taste of noodle products. Attached Figure Description
[0022] Figure 1 The image shows the results of syringic acid production after fermentation by strain XJ0805.
[0023] Figure 2 The image shows the detection results of butyl hexanoate produced by strain XJ0805 after fermentation.
[0024] Figure 3 This is a morphological observation of strain XJ0805 on MRS solid medium.
[0025] Figure 4 This is a microscopic observation of strain XJ0805 after Gram staining. Detailed Implementation
[0026] The present invention will be further described below with reference to specific embodiments:
[0027] Unless otherwise specified, the experimental methods used in the following implementation examples are all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.
[0028] Example 1, see attached document Figure 1-4 A strain of *Lactobacillus paracasei* that improves the flavor of whole-grain sourdough.
[0029] The strain is Lacticaseibacillus paracasei INM3110, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCCNO:64515.
[0030] A strain of *Lactobacillus paracasei* that improves the flavor of whole-grain sourdough, wherein the colony characteristics of *Lactobacillus paracasei* are as follows: after 48 h of culture on MRS solid medium, the colonies are raised, round, smooth, dense, and white.
[0031] Example 2, a method for preparing *Lactobacillus paracasei* to improve the flavor of whole grain sourdough, as follows:
[0032] 1. Raw material processing
[0033] Take the kimchi juice and dilute it with sterile saline solution 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 containing 0.015% bromocresol purple. Incubate at 37°C for 48 h. Select single colonies that are yellow, 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 XJ0801 to XJ0809.
[0034] 2. Preliminary screening of bacterial strains
[0035] The nine bacterial strains were inoculated into MRS liquid medium at a 1% inoculum concentration and cultured at 37°C for 24 hours. Then, they were inoculated into induction culture medium at a 1% inoculum concentration and cultured statically at 37°C for 24 hours, until the final bacterial density reached 10⁻⁶. 6 cfu / mL.
[0036] After 24 hours of fermentation, samples were taken from each tube and centrifuged at 10,000 rpm for 10 minutes. The supernatant obtained was the sample, which was then used for physicochemical analysis.
[0037] Induction culture medium: mulberry juice, sterilized at 60℃ for 30 minutes.
[0038] Syringic acid detection: High-performance liquid chromatography (HPLC) was used. Mobile phase: methanol:water:36% acetic acid = 40 / 60 / 3; flow rate: 1 mL / min; detection wavelength: 272 nm; column temperature: 30℃; chromatographic column: CAPCELL PAK ADME (4.6 × 250 mm); sample loading volume: 10 μL (sample and standard dissolved in ethanol). Figure 1 The results are for the detection of syringic acid produced after fermentation of XJ0805.
[0039] Butyl hexanoate detection: A DB-WAX (30m, 0.25mm, 0.25μm) capillary column was used. GC conditions were: helium as carrier gas, carrier gas flow rate set to 1 mL / min, splitless injection, and injection port temperature of 250℃. MS conditions were: ion source temperature 230℃, MS quadrupole temperature 150℃, electron energy 70 eV, mass spectrometry scan range 45–500, and full scan mode. The column oven temperature program was: 40℃ for 2 min, increased to 150℃ at 3℃ / min, then increased to 220℃ at 20℃ / min and held for 1 min. Compound identification was performed using a mass spectrometry database method. Each peak was compared to the NIST Library mass spectrometry database by computer; a match greater than 800 was used as the qualitative criterion. Figure 2 The results are for the detection of butyl hexanoate produced after fermentation of XJ0805.
[0040] Table 1. Analysis of total acid, eugenol, and butyl hexanoate produced during fermentation.
[0041]
[0042] Strains XJ0802, XJ0804, and XJ0805, which had high contents of total acid, syringic acid, and butyl hexanoate, were selected for secondary screening.
[0043] 3. Secondary screening of strains
[0044] The initially screened lactic acid bacteria strains XJ0802, XJ0804, and XJ0805 were reactivated and cultured at a cell concentration of 10. 6The starter culture was prepared according to the national standard GB / T14612-2008, and whole grain bread was made from it. Sensory evaluation was performed, and the results are shown in Table 2.
[0045] Table 2 Sensory Analysis of Fermented Bread
[0046] XJ0802 4.5 4.5 4.5 XJ0804 5.0 4.5 4.75 XJ0805 5.0 5.0 5.0
[0047] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.
[0048] As shown in Table 2, XJ0805 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 3 As shown, the colonies are round, smooth, dense, milky white, and 1.8-2.0 mm in size; the results are shown in the figure below. Figure 3 As shown. Under a microscope, XJ0805 appears as rod-shaped, paired, or short chains, with individual morphologies measuring 0.5-1.0 μm in width and 2.0-10.0 μm in length, and does not form spores. The results are as follows. Figure 4 As shown.
[0049] The XJ0805 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 *Lactaseibacillus paracasei*, confirming that this strain is a *Lactaseibacillus paracasei* strain that improves the flavor of whole-grain sourdough and can be applied in the food fermentation industry. It was named *Lactaseibacillus paracasei* INM3110. This strain was deposited on April 11, 2024, at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCC No. 64515, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Therefore, in this application, INM3110 and XJ0805 refer to the same strain. The gene sequence of the strain INM3110 of this invention is as follows:
[0050]
[0051] Example 3: Detection of the genetic stability of Lactobacillus paracasei INM3110
[0052] The *Lactobacillus paracasei* INM3110 from Example 2 was passaged 10 times on MRS liquid medium. The samples from the 1st, 5th, and 10th generations were centrifuged at 10,000 rpm for 10 min, and the supernatant was used as the sample. Physicochemical indicators were analyzed, and the methods for determining syringic acid and butyl hexanoate content were the same as in Example 2. Sensory evaluation was also performed to assess its passage stability; specific data are shown in Table 3.
[0053] Table 3. Results of the passaging stability test
[0054] INM3110 1st generation 43.32±0.15 39.54±0.21 5.0 INM3110 5th generation 41.28±0.26 38.72±0.14 5.0 INM3110 10th generation 42.64±0.31 39.41±0.27 5.0
[0055] Table 3 shows that the fermentation performance of Lactobacillus paracasei INM3110 strains of different generations was normal, and the content of eugenol and butyl hexanoate varied within 10%. The strains showed good genetic stability and met the requirements for production and use.
[0056] Example 4: Bread making using Lactobacillus paracasei INM3110 sourdough starter.
[0057] Lactobacillus paracasei INM3110 was inoculated into a fermented sourdough starter to produce whole grain sourdough starter and whole grain bread according to the national standard GB / T14612-2008. The concentration of the fermentation bacteria was 10... 4 -10 8 At CFU / mL, whole grain bread was prepared separately, with XJ0809 as the control strain. The method for detecting the content of eugenol and butyl hexanoate in the sourdough strain is the same as in Example 2. The results are shown in Table 4.
[0058] Table 4. Content of eugenol and butyl hexanoate in whole grain sourdough starter
[0059]
[0060] Table 5 Sensory evaluation results of whole grain bread
[0061]
[0062]
[0063] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores.
[0064] The results in Tables 4 and 5 show that *Lactobacillus paracasei* INM3110 significantly increases the content of eugenol and butyl hexanoate in the sourdough starter in whole-grain bread, and the higher the inoculum size, the higher the content of eugenol and butyl hexanoate. However, excessively high or low inoculum concentrations of *Lactobacillus paracasei* INM3110 affect the sourness, sweetness, and texture of the bread. The optimal inoculum concentration is 1.0 × 10⁻⁶. 7 CFU / mL, this concentration produces sufficient eugenol and butyl hexanoate, while also enhancing the flavor and texture of whole-grain bread.
[0065] Example 5: Making baguettes using Lactobacillus paracasei INM3110 sourdough starter.
[0066] The final concentration of Lactobacillus paracasei INM3110 was 10. 7 The whole grain sourdough inoculum concentration was prepared using a dose of CFU / mL. Another group was inoculated with XJ0809 as a control group. Bagels were then prepared according to the national standard GB / T14612-2008.
[0067] After fermentation, the eugenol and butyl hexanoate in the whole grain baguette were tested according to the method in Experiment Example 2. 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.
[0068] Table 6. Results of Whole Grain Sourdough Detection and Sensory Evaluation of Baguettes
[0069]
[0070]
[0071] 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.
[0072] As shown in Table 6, the results indicate that whole grains were inoculated for 10 days... 7 Using CFU / mL Lactobacillus paracasei INM3110 to make sourdough starter can significantly increase the content of eugenol and butyl hexanoate in 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.
[0073] Example 6: Making croissants using Lactobacillus paracasei INM3110 sourdough starter
[0074] The final concentration of Lactobacillus paracasei INM3110 was 10. 7 The whole grain noodle starter concentration was prepared using a dose of CFU / mL. Another group was inoculated with XJ0809 as a control group. Croissants were then prepared according to the national standard GB / T14612-2008.
[0075] After fermentation, the content of eugenol and butyl hexanoate in the whole grain sourdough starter was tested according to the method in Experiment Example 2. 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.
[0076] Table 7. Results of Whole Grain Pasta Starter Testing and Sensory Evaluation of Croissants
[0077]
[0078] 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.
[0079] As shown in Table 7, inoculating whole grains with 10 7 Using CFU / mL Lactobacillus paracasei INM3110 to make sourdough starter can significantly increase the content of eugenol and butyl hexanoate in fermented starter; in addition, it can improve the sensory evaluation of croissants, increase the aroma of croissants, enhance the flavor and texture of croissants, and make croissants fragrant and sweet and sour.
[0080] Example 7: Bagel preparation using Lactobacillus paracasei INM3110 starter.
[0081] The final concentration of Lactobacillus paracasei INM3110 was 10. 7 The whole grain sourdough inoculum concentration was prepared using a dose of CFU / mL. Another group was inoculated with XJ0809 as a control group. Bagels were then prepared according to the national standard GB / T14612-2008.
[0082] After fermentation, the content of eugenol and butyl hexanoate in the whole grain sourdough starter was tested according to the method in Experiment Example 2. 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 7 Using CFU / mL Lactobacillus paracasei INM3110 to make sourdough starter can significantly increase the content of eugenol and butyl hexanoate in the fermented starter; in addition, it can improve the sensory evaluation of bagels, increase bagel aroma, enhance bagel flavor and texture, and make bagels fragrant, sweet and sour and delicious.
[0087] Based on the results of the above embodiments, the Lacticaseibacillus paracasei INM3110 provided by the present invention, when applied to the fermentation of whole grain sourdough starter, can significantly increase the content of eugenol and butyl hexanoate in 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 wheat products.
[0088] Application of a strain of *Lactobacillus paracasei* that effectively enhances the production of higher concentrations of eugenol and butyl hexanoate after fermentation of sourdough.
[0089] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, which fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention patent should be determined by the appended claims.
[0090]
[0091]
Claims
1. A strain of *Lactobacillus paracasei* that improves the flavor of whole-grain sourdough, characterized by: The strain is Lacticaseibacillus paracasei INM3110, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCC NO:64515.
2. The *Lactobacillus paracasei* strain for improving the flavor of whole-grain sourdough according to claim 1, characterized in that: The colony characteristics of *Lactobacillus paracasei* are as follows: after culturing on MRS solid medium for 48 hours, the colonies are raised, round, smooth, dense, and white.
3. The application of the *Lactobacillus paracasei* strain for improving the flavor of whole-grain sourdough as described in claim 1, characterized in that: According to the production process of whole grain products, the fermentation whole grain liquid has 10 4 -10 8 cfu / mL of Paracasei subsp. paracasei, the fermentation time is 3d, and the fermentation temperature is 37℃.
4. The application of the *Lactobacillus paracasei* strain for improving the flavor of whole-grain sourdough as described in claim 1, characterized in that: It effectively increases the concentration of eugenol and butyl hexanoate after fermentation of sourdough.