Preparation and application of *Lactobacillus plantarum* INM3104 strain that reduces raffinose in whole grain dough

Abstract

This invention provides the preparation and application of a strain of *Lactiplantibacillus plantarum* INM3104 that reduces raffinose in whole grain dough. This strain, *Lactiplantibacillus plantarum* INM3104, which degrades raffinose, was deposited at the Guangdong Provincial Microbial Culture Collection Center on April 11, 2024, with accession number GDMCC NO:64512. The advantages are: it only reduces the raffinose content in whole grain products, while also improving the flavor and quality of the whole grain products; the whole grain products produced using this preparation do not affect the overall processing of the whole grain products after fermentation, not only improving the taste of the whole grain products but also reducing the adverse factors associated with whole grain products.

Description

Technical Field

[0001] This invention relates to the preparation and application of a strain of Lactobacillus plantarum INM3104 suitable for reducing raffinose in whole grain dough during the fermentation stage of whole grain sourdough starter. Background Technology

[0002] Raffinose (also known as cottonseed sugar or raffinose trisaccharide, with the structural formula α-D-galactose-α-D-glucose-β-D-fructose) is widely found in plants in nature. It is present in varying amounts in many vegetables (cabbage, cauliflower, potatoes, beets, onions, etc.), fruits (grapes, bananas, kiwis, etc.), grains (wheat, rice, oats, etc.), and the kernels of some oilseed crops (soybeans, sunflower seeds, cottonseeds, peanuts, etc.). The human digestive tract lacks the enzyme to hydrolyze and utilize raffinose. Once in the colon, raffinose is fermented and utilized by gas-producing bacteria, producing intestinal gas. This is why eating grains and legumes (rich in raffinose) can cause intestinal gas.

[0003] Raffinose has a strong ability to promote the growth of beneficial bacteria such as Bifidobacteria and Lactobacillus, while effectively inhibiting the growth of harmful bacteria in the intestines, thus establishing a healthy intestinal flora environment. Therefore, utilizing lactic acid bacteria metabolism can not only effectively reduce raffinose but also increase the content of lactic acid bacteria in the human body.

[0004] Lactobacillus plantarum (formerly known as Lactobacillus plantarum) is a type of lactic acid bacteria. Its optimal growth temperature is 30℃. It is anaerobic or facultative anaerobic. The bacteria are straight or curved rods, single, sometimes in pairs or chains. The optimal pH is around 6.5. It belongs to homofermentative lactic acid bacteria. Lactobacillus plantarum has many health benefits, including: (1) a certain immunomodulatory effect; (2) an inhibitory effect on pathogenic bacteria; (3) reducing serum cholesterol levels and preventing cardiovascular diseases; (4) maintaining the balance of intestinal flora; (5) promoting the absorption of nutrients; (6) alleviating lactose intolerance; (7) inhibiting the formation of tumor cells, etc. At present, there is little research on the metabolism of raffinose by lactic acid bacteria. Patent: A short lactobacillus that can ferment soybean oligosaccharides and its application - CN202310260013.1. This invention relates to a short lactobacillus that can effectively reduce raffinose in soybeans and produce a large amount of organic acids. Patent: A strain of *Lactobacillus harzianum* capable of fermenting soybean oligosaccharides and its application - CN202211005378.1. This invention also relates to a strain of *Lactobacillus harzianum* capable of effectively degrading raffinose in soybeans. However, the lactic acid bacteria involved in these two patents are not included in the "List of Edible Fungi," and therefore cannot be effectively applied in actual production and processing. Patent: Process for producing highly active soybean oligosaccharides by lactic acid bacteria fermentation - CN200610045808.7. This patent involves lactic acid bacteria that cannot effectively degrade raffinose, and therefore has a different application direction from this patent.

[0005] The above patents all focus on using lactic acid bacteria to reduce raffinose. Although these lactic acid bacteria possess the ability to degrade raffinose, they are not included in the national list of strains permitted for use. Therefore, developing a safe and effective strain for reducing raffinose has broad application prospects.

[0006] Although many studies have shown that there are effective methods for degrading raffinose content, there are still shortcomings in practical applications: (1) The microorganisms involved in the biochemical degradation of raffinose are not on the list of edible fungi and cannot be applied to food. (2) In industrial applications, there is still a lack of lactic acid bacteria that can be directly applied to degrade raffinose during the whole grain fermentation stage. Summary of the Invention

[0007] To address the above problems, this invention provides a method for preparing and applying Lactobacillus plantarum INM3104, a strain that reduces raffinose in whole grain dough. Without altering existing processes, this method involves fermenting whole grain sourdough starter with Lactobacillus plantarum to produce whole grain products with reduced raffinose.

[0008] The objective of this invention is achieved through the following method: a strain of *Lactobacillus plantarum* that reduces raffinose in whole grain dough.

[0009] The strain is Lactiplantibacillus plantarum INM3104, which degrades raffinose. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCC NO:64512.

[0010] Furthermore, the colony characteristics of the *Lactobacillus plantarum* that degrades raffinose are as follows: after culturing on MRS solid medium for 48 hours, the colony diameter reaches 2.5–3.5 mm, the colonies are raised, round, smooth, dense, and white.

[0011] A method for preparing *Lactobacillus plantarum* that reduces raffinose in whole grain dough is as follows:

[0012] (1) Isolation: Lactic acid bacteria are isolated and screened from the cream. The cream product is processed into a paste and added to sterile water to dilute to a concentration of 10. 5 -10 6 Different concentrations of dairy products were evenly spread on MRS solid medium containing 0.015% bromocresol purple and incubated at 37°C for 48 hours. Yellow colonies and lactic acid bacteria with the correct morphology were selected from the MRS solid medium for screening.

[0013] (2) Screening: The obtained lactic acid bacteria were cultured to the logarithmic growth phase, and then inoculated into the induction liquid medium with an inoculation amount of 1% and a final strain concentration of 1×106cfu / mL. After being placed in a 37℃ static culture for 2 days, the fermentation broth was obtained; the raffinose content was measured and selected.

[0014] Furthermore, the formulation of the induction liquid culture medium is as follows: weigh 4g yeast extract powder, 5g raffinose, and 0.04g manganese sulfate, dissolve them in 1000mL distilled water, and sterilize at 0.1MPa and 121℃ for 15min.

[0015] Furthermore, according to the production process of whole grain products, the inoculum quantity in the fermented whole grain liquid is 10. 4 -10 8 Lactobacillus plantarum at cfu / mL was fermented for 3 days at a temperature of 37℃.

[0016] Application of a strain of *Lactobacillus plantarum* that effectively reduces the high concentration of raffinose produced after fermentation of whole-grain sourdough.

[0017] Advantages of the present invention: (1) The screening method is universal and can be widely used to screen strains that reduce the raffinose content in whole grain products.

[0018] (2) It can enhance the flavor of whole grains, and there will be 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.

[0019] (3) It can effectively reduce the high concentration of raffinose produced after fermentation of whole grain sourdough.

[0020] (4) It not only reduces the content of raffinose in whole grain products, but also improves the flavor and quality of whole grain products. The whole grain products produced by applying this preparation will not affect the overall process of whole grain products after fermentation. It not only improves the taste of whole grain products, but also reduces the adverse factors brought by whole grain products. Attached Figure Description

[0021] Figure 1 Comparison of NM0802 strain inoculated with induction liquid medium.

[0022] Figure 2 This is a morphological observation of strain NM0802 on MRS solid medium.

[0023] Figure 3 This is a microscopic image of strain NM0802 after Gram staining. 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 all conventional methods; the materials and reagents used are all commercially available unless otherwise specified.

[0026] Example 1, see attached document Figure 1-3 A strain of *Lactobacillus plantarum* that reduces raffinose in whole-grain dough.

[0027] The strain is Lactiplantibacillus plantarum INM3104, which degrades raffinose. It was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCC NO:64512.

[0028] A strain of *Lactobacillus plantarum* that reduces raffinose in whole grain dough. The colony characteristics of the *Lactobacillus plantarum* that degrades raffinose are as follows: after 48 hours of culture on MRS solid medium, the colonies are raised, round, smooth, dense, and white.

[0029] Example 2, a method for preparing *Lactobacillus plantarum* that reduces raffinose in whole grain dough, 1. Raw material processing

[0030] Take the cream 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 (containing 0.015% bromocresol purple) plates. 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 NM0801 to NM0809.

[0031] 2. Preliminary screening of bacterial strains

[0032] 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 liquid 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. Results are as follows: Figure 1 .

[0033] After 24 hours of fermentation, samples were taken from each tube, centrifuged at 10,000 r / min for 10 min, and the supernatant was obtained as the sample for physicochemical index testing.

[0034] Induction liquid culture medium: Weigh 4g yeast extract powder, 5g raffinose, 0.04g manganese sulfate, and 1g Tween 80, dissolve them in 1000mL distilled water, and sterilize at 0.1MPa and 121℃ for 15min.

[0035] Raffinose detection: ELISA reagents were used, and the procedure was followed according to the instructions. Results are shown in Table 1.

[0036] Table 1. Analysis of total acid and raffinose produced during fermentation.

[0037]

[0038]

[0039] Strains NM0802, NM0805, and NM0807, which had high total acid and raffinose content, were selected for secondary screening.

[0040] 3. Secondary screening of strains

[0041] The initially screened lactic acid bacteria strains NM0802, NM0805, and NM0807 were reactivated and cultured at a cell concentration of 10. 6 The 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.

[0042] Table 2 Sensory Analysis of Fermented Bread

[0043] strain name Bread appearance taste Average score NM0802 4.8 5.0 4.9 NM0805 4.8 4.8 4.8 NM0807 5.0 4.5 4.75

[0044] Note: Each indicator has a maximum score of 5 points, and the higher the score, the better the result.

[0045] As shown in Table 2, NM0802 had the best sensory evaluation results. As a target strain for secondary screening, the morphological observation of this strain on MRS solid medium was as follows: Figure 2 As shown, colonies are round, smooth, dense, and milky white, 1.8-2.0 mm in diameter; 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. Results are as follows... Figure 3 As shown.

[0046] The NM0802 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 100% similarity to *Lactiplantibacillus plantarum*, confirming that this strain is indeed *Lactiplantibacillus plantarum* and can be used in the food fermentation industry. It was named *Lactiplantibacillus plantarum* INM3104. This strain was deposited on April 11, 2024, at the Guangdong Provincial Microbial Culture Collection Center, accession number GDMCCNo. 64512, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province. Therefore, in this application, INM3104 and NM0802 refer to the same strain. The gene sequence of the strain INM3104 of this invention is as follows:

[0047]

[0048] Example 3: Detection of the genetic stability of Lactobacillus plantarum INM3104

[0049] The *Lactobacillus plantarum* INM3104 from Example 1 was passaged 10 times in inoculation induction 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 detecting total acid content and raffinose degradation rate were the same as in Example 2. Sensory evaluation was also performed to determine its passage stability; specific data are shown in Table 3.

[0050] Table 3. Results of the passaging stability test

[0051]

[0052]

[0053] Table 3 shows that the fermentation performance of Lactobacillus plantarum INM3104 strains of different generations was normal, with the total acid and raffinose content varying within 10%, indicating good genetic stability and meeting the requirements for production and use.

[0054] Example 4: Bread made with Lactobacillus plantarum INM3104 whole wheat sourdough starter.

[0055] Lactobacillus plantarum INM3104 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 NM0809 as the control strain. The method for detecting the total acid and raffinose degradation rate in the sourdough strain was the same as in Example 2, and the results are shown in Table 4.

[0056] Table 4. Total acid content and raffinose degradation rate in whole-grain sourdough starter

[0057]

[0058] Table 5 Sensory evaluation results of whole grain bread

[0059] Grouping Inoculation volume sour sweet taste Overall score Control group 1 <![CDATA[1.0×10 4 ]]> 7.2 6.5 7.7 21.4 Experimental group 1 <![CDATA[1.0×10 4 ]]> 6.8 7.2 8.5 22.5 Control group 2 <![CDATA[1.0×10 5 ]]> 6.3 7.6 9.2 23.1 Experimental group 2 <![CDATA[1.0×10 5 ]]> 6.6 8.2 8.5 23.3 Control group 3 <![CDATA[1.0×10 6 ]]> 7.5 8.3 7.2 23.0 Experimental group 3 <![CDATA[1.0×10 6 ]]> 9.2 8.9 9.2 27.3 Control group 4 <![CDATA[1.0×10 7 ]]> 6.6 6.4 8.0 21.0 Experimental group 4 <![CDATA[1.0×10 7 ]]> 9.4 9.7 9.6 28.7 Control group 5 <![CDATA[1.0×10 8 ]]> 6.6 7.7 8.4 22.7 Experimental group 5 <![CDATA[1.0×10 8 ]]> 3.2 5.4 7.3 15.9

[0060] Note: Each assessment item is worth 10 points, with a scoring interval of 0.1. The overall score is the sum of all scores.

[0061] As shown in Tables 4 and 5, *Lactobacillus plantarum* INM3104 significantly increased the total acid content and raffinose degradation rate of the sourdough starter in whole-grain bread. Furthermore, the higher the inoculum concentration, the higher the raffinose degradation rate and total acid content. However, excessively high or low inoculum concentrations of *Lactobacillus plantarum* INM3104 affected the sourness, sweetness, and texture of the bread. 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.

[0062] Example 5: Making croissants using Lactobacillus plantarum INM3104 sourdough starter.

[0063] The final concentration of *Lactobacillus plantarum* INM3104 was 10. 7 The whole grain sourdough starter concentration was prepared using a dose of CFU / mL. Another group was inoculated with NM0809 as a control group. Croissants were then prepared according to the national standard GB / T14612-2008.

[0064] After fermentation, the total acid content and raffinose degradation rate of the whole grain croissant 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 flavor of the croissant. The results are recorded in Table 6.

[0065] Table 6. Results of Whole Grain Sourdough Detection and Sensory Evaluation of Croissants

[0066]

[0067] 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.

[0068] As shown in Table 6, the results indicate that whole grains were inoculated for 10 days... 7 Using CFU / mL Lactobacillus plantarum INM3104 to make sourdough starter can significantly increase the degradation rate of raffinose. In addition, the total acid content of fermented sourdough starter can improve the sensory evaluation of croissants, increase their aroma, enhance their flavor and texture, and make them fragrant, sweet and sour.

[0069] Example 6: Making soft bread using Lactobacillus plantarum INM3104 whole grain sourdough starter.

[0070] The final concentration of *Lactobacillus plantarum* INM3104 was 10. 7 The whole grain starter culture concentration was prepared using a dose of CFU / mL. Another group was inoculated with NM0809 as a control group. Soft bread was then prepared according to the national standard GB / T14612-2008.

[0071] After fermentation, the total acid content and raffinose content of the whole grain sourdough starter 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 flavor of the soft bread. The results are recorded in Table 7.

[0072] Table 7. Results of whole grain starter testing and sensory evaluation of soft bread.

[0073]

[0074] 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.

[0075] As shown in Table 7, inoculating whole grains with 10 7 Using CFU / mL Lactobacillus plantarum INM3104 to make sourdough starter can significantly increase the degradation rate of raffinose. In addition, the total acid content can improve the sensory evaluation of soft bread, increase the aroma of soft bread, and enhance the flavor and texture of soft bread, making it fragrant and delicious.

[0076] Example 7: Bagel preparation using Lactobacillus plantarum INM3104 starter.

[0077] The final concentration of *Lactobacillus plantarum* INM3104 was 10. 7 The whole grain sourdough inoculum concentration was prepared using a dose of CFU / mL. Another group was inoculated with NM0809 as a control group. Bagels were then prepared according to the national standard GB / T14612-2008.

[0078] After fermentation, the total acid content and raffinose degradation rate of the whole grain sourdough starter 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 flavor of the soft bread. The results are recorded in Table 8.

[0079] Table 8 Results of Whole Grain Sourdough Detection and Bagel Sensory Evaluation

[0080]

[0081] 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.

[0082] As shown in Table 8, inoculating whole grains with 10 7 Using CFU / mL Lactobacillus plantarum INM3104 to make sourdough starter can significantly increase the degradation rate of raffinose; in addition, the total acid content of fermented sourdough starter can also improve the sensory evaluation of bagels, increase bagel aroma, and enhance bagel flavor and texture, making bagels fragrant, sweet and sour and delicious.

[0083] Based on the results of the above embodiments, the Lactiplantibacillus plantarum INM3104 provided by the present invention, when applied to the fermentation of whole grain sourdough starter, can significantly improve the degradation rate of raffinose in whole grains without changing the existing process. At the same time, the total acid content can also improve the aroma and taste of whole grain products, significantly enhancing the quality of whole grain products.

[0084] Application of a strain of *Lactobacillus plantarum* that effectively reduces the high concentration of raffinose produced after fermentation of whole-grain sourdough.

[0085] 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.

[0086]

[0087]

Claims

1. A strain of *Lactobacillus plantarum* that reduces raffinose in whole grain dough, characterized by: This strain is *Lactobacillus plantarum*, which degrades raffinose. Lactiplantibacillus plantarum INM3104 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on April 11, 2024, with accession number GDMCC NO:64512.

2. The *Lactobacillus plantarum* strain that reduces raffinose in whole grain dough according to claim 1, characterized in that: The colony characteristics of *Lactobacillus plantarum* that degrades raffinose are as follows: after 48 hours of culture on MRS solid medium, the colonies are raised, round, smooth, dense, and white.

3. The application of the *Lactobacillus plantarum* strain that reduces raffinose in whole grain dough as described in claim 1, characterized in that: According to the production process of whole grain products, the inoculum quantity in the fermented whole grain liquid is 10. 4 -10 8 Lactobacillus plantarum at cfu / mL was fermented for 3 days at a temperature of 37℃.

4. The application of the *Lactobacillus plantarum* strain as described in claim 1, which effectively reduces the production of high concentrations of raffinose after fermentation of whole-grain sourdough, is characterized in that: Inoculate 10g of whole grains 7 Using CFU / mL Lactobacillus plantarum INM3104 to prepare sourdough starter can significantly increase the degradation rate of raffinose.

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