A method for increasing the folate content of bread

Abstract

The application belongs to the technical field of biology and specifically relates to a method for increasing the content of folic acid in bread. The method specifically comprises the following steps: light-proof germination of black amaranth seeds, drying and crushing to obtain germinated powder; mixing the germinated powder, Lactobacillus plantarum powder, rice bran fatty alkanol and the like, and then standing and culturing to obtain an acid dough; mixing the acid dough with high-gluten flour, active dry yeast and sedoheptulose, stirring, adding butter, and then twice stirring, fermentation, division and baking to obtain the finished product. The test results show that the addition of rice bran fatty alkanol can promote the proliferation of Lactobacillus plantarum, and significantly increase the natural folic acid content of the bread; at the same time, the replacement of sucrose with sedoheptulose can improve the specific volume and texture characteristics of the bread, realize the efficient enrichment of natural folic acid, and the product has excellent taste and low-sugar health attributes, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for increasing the folic acid content in bread. Background Technology

[0002] Folic acid (vitamin B9), an important coenzyme in the one-carbon unit transferase system, is an essential water-soluble vitamin that plays a crucial role in cell division, growth, and amino acid metabolism. The human body cannot synthesize it and must obtain it through diet. Folic acid deficiency can lead to megaloblastic anemia, hyperhomocysteinemia, and increase the risk of neural tube defects and cardiovascular disease. Therefore, the development of folic acid-fortified foods is of great significance.

[0003] Currently, the main approaches to folic acid fortification in food include direct addition of chemically synthesized folic acid and bioaccumulation of natural folic acid. While chemically synthesized folic acid is inexpensive, excessive long-term intake may mask vitamin B12 deficiency symptoms, and its bioavailability and metabolic pathways differ from natural folic acid. In contrast, bioaccumulation of natural folic acid through fermentation offers higher bioactivity and safety, and has become an important development trend. Lactic acid bacteria are highly favored due to their GRAS (Generally Recognized As Safe) properties and metabolic plasticity; some lactic acid bacteria possess the ability to synthesize folic acid de novo. However, their synthesis efficiency in dough is limited by factors such as strain characteristics and nutrient substrates, resulting in limited practical application effects.

[0004] Quinoa seeds can activate endogenous folate synthesis and metabolism during germination, significantly increasing folate content and providing a new approach for the development of high-folate food ingredients. However, current technologies have not yet achieved a synergistic effect between folate enrichment during quinoa germination and lactic acid bacteria fermentation. Furthermore, traditional bread uses sucrose as the fermentation carbon source, which is insufficient to meet the needs of people controlling their blood sugar levels. While sedum heptulose has low calorie and low glycemic response characteristics, its use as a substitute for sucrose in the preparation of high-folate bread has not yet been reported.

[0005] In summary, developing a method for preparing high-folate bread that combines quinoa biogermination, lactic acid bacteria fermentation, and functional sugar substitution is of great practical significance for improving the nutritional added value of baked goods and meeting the health needs of specific populations. Summary of the Invention

[0006] The purpose of this invention is to provide a method for increasing the folic acid content in bread.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A method for increasing the folic acid content in bread, the method specifically includes the following steps: (1) After washing the black quinoa seeds, soak them in water and germinate them in the dark; after germination, dry them, crush them and sieve them to obtain germinated quinoa powder; (2) By weight, take 8-12 parts of sprouted quinoa powder, 1-2 parts of Lactobacillus plantarum powder, 0.01-0.03 parts of rice bran fatty alkyl alcohol, 24-30 parts of water and 14-16 parts of high gluten wheat flour obtained in step (1), mix them, and let them stand to cultivate to obtain sour dough; (3) By weight, take 25-30 parts of sourdough, 35-40 parts of high-gluten flour, 0.6-1 parts of active dry yeast, 1-2 parts of sedum heptose, 0.4-0.6 parts of salt and 25-30 parts of water obtained in step (2), mix them, stir at low speed to form a dough, and then stir at high speed to obtain premixed ingredients. (4) By weight, take 45-50 parts of premixed ingredients obtained in step (3) and 1-2 parts of butter to make a mixed dough. Mix at low speed twice, mix at high speed twice, and let it stand to ferment to obtain the first fermented dough. (5) Take the first fermented dough obtained in step (4), ferment it a second time, divide it, and obtain bread dough; (6) Take the bread dough obtained in step (5) and bake it to obtain natural high folic acid bread.

[0008] Furthermore, in step (1), the temperature for germination in the dark is 20-25℃, the relative humidity is 85%-90%, and the time is 32-42 h; the drying temperature is 45-55℃; the drying is carried out until the moisture content is <10wt%, and the sieve mesh size is 80-100 mesh.

[0009] Furthermore, in step (2), the static culture temperature is 28-32℃, and the static culture time is 16-20h; the viable count of the *Lactobacillus plantarum* powder is 5-7 × 10⁻⁶. 7 CFU / g.

[0010] Furthermore, in step (3), the low-speed stirring time is 2-3 minutes and the low-speed stirring speed is 130-140 rpm; the high-speed stirring time is 3-4 minutes and the high-speed stirring speed is 200-220 rpm.

[0011] Furthermore, in step (4), the dough is allowed to ferment until it doubles in volume, and the fermentation temperature is 25-30°C; the low-speed mixing time is 2-3 minutes, and the low-speed mixing speed is 130-140 rpm; the high-speed mixing time is 3-4 minutes, and the high-speed mixing speed is 200-220 rpm.

[0012] Furthermore, in step (5), the secondary fermentation is carried out to 1.5 times the volume of the initial fermented dough, and the temperature of the secondary fermentation is 35-38℃.

[0013] Furthermore, in step (6), the upper heat temperature is 180–190℃ and the lower heat temperature is 170–180℃; the baking time is 20–30 min.

[0014] Compared with the prior art, the main advantages of the present invention are: (1) This invention utilizes biogermination technology to activate the endogenous folic acid synthesis metabolism of quinoa seeds, significantly increasing the folic acid content of the raw material base. On this basis, Lactobacillus plantarum and rice bran fatty alkanol are combined for use in the sourdough fermentation system. Rice bran fatty alkanol can effectively promote the growth activity of Lactobacillus plantarum in the dough, thereby significantly enhancing its ability to biosynthesize folic acid. The combination of Lactobacillus plantarum and quinoa seeds significantly increases the folic acid content in the final bread, which can efficiently supplement the human body's folic acid level, has good nutritional value and functionality, is green and environmentally friendly, and is easy to industrialize.

[0015] (2) Replacing sucrose with sedum heptose results in bread with a larger internal volume, lower hardness, and better chewiness, as well as a richer flavor. In addition, sedum heptose has the advantages of low calories and low glycemic response, providing a new bread option for consumers who need to control their sugar intake. Attached Figure Description

[0016] Figure 1 The effect of fatty alkyl alcohols in rice bran on the viable count of Lactobacillus plantarum during the fermentation of sourdough. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to specific embodiments. However, those skilled in the art should understand that the following embodiments are only for illustrating the present invention and should not be regarded as limiting the present invention. Specific conditions not specified in the embodiments are performed according to conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the reagents or instruments used are all conventional products obtained through commercial channels.

[0018] The Lactobacillus plantarum powder used in the embodiments of this invention was purchased from Microcare Probiotics Co., Ltd.

[0019] Example 1 A method for increasing the folic acid content in bread, the method specifically includes the following steps: (1) After washing the black quinoa seeds, soak them in water and germinate them in the dark for 37 h at a temperature of 22℃ and a relative humidity of 87%. After germination, remove the moisture and dry them at 50℃ until the moisture content is <10wt%. Then, crush them through a 90-mesh sieve to obtain germinated quinoa powder.

[0020] (2) In parts by weight, 10 parts of germinated quinoa flour obtained in step (1), 1 part of Lactobacillus plantarum powder, 0.03 part of rice bran fatty alcohol, 21 parts of water and 11 parts of high-gluten wheat flour are mixed, and statically cultured at 30°C for 18 h to obtain a sourdough; the viable count of the Lactobacillus plantarum powder is 6×10 7 CFU / g.

[0021] (3) In parts by weight, 27 parts of the sourdough obtained in step (2), 37 parts of high-gluten flour, 0.8 part of active dry yeast, 1.5 parts of sedoheptulose, 0.5 part of table salt and 27 parts of water are mixed, stirred at a low speed of 135 rpm for 2 min to form a dough, and then stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material; (4) In parts by weight, 47 parts of the premixed raw material obtained in step (3) and 1 part of butter are mixed to obtain a mixed dough, which is stirred at a secondary low speed of 135 rpm for 2 min, stirred at a secondary high speed of 210 rpm for 3 min, and subjected to static fermentation at 27°C until the volume of the mixed dough doubles, to obtain a primary fermented dough; (5) The primary fermented dough obtained in step (4) is subjected to secondary fermentation at 36°C until the volume of the primary fermented dough reaches 1.5 times, and then divided to obtain bread dough; (6) The bread dough obtained in step (5) is baked at an upper heating temperature of 185°C and a lower heating temperature of 175°C for 25 min, to obtain the natural high folate bread.

[0022] Example 2 A method for increasing the folate content in bread, the method specifically comprises the following steps: (1) Black quinoa seeds are cleaned, then wetted and soaked, and subjected to light-shielded germination for 32 h under the conditions of a temperature of 20°C and a relative humidity of 85%; after germination, the seeds are dehumidified at 45°C, dried at 45°C until the moisture content is less than 10 wt%, and pulverized and sieved through an 80-mesh sieve to obtain germinated quinoa flour.

[0023] (2) In parts by weight, 8 parts of germinated quinoa flour obtained in step (1), 1 part of Lactobacillus plantarum powder, 0.02 part of rice bran fatty alcohol, 18 parts of water and 10 parts of high-gluten wheat flour are mixed, and statically cultured at 28°C for 16 h to obtain a sourdough; the viable count of the Lactobacillus plantarum powder is 5×10 7 CFU / g.

[0024] (3) In parts by weight, 25 parts of the sourdough obtained in step (2), 35 parts of high-gluten flour, 0.6 part of active dry yeast, 1 part of sedoheptulose, 0.4 part of table salt and 25 parts of water are mixed, stirred at a low speed of 130 rpm for 2 min to form a dough, and then stirred at a high speed of 200 rpm for 3 min to obtain a premixed raw material; (4) In parts by weight, 45 parts of the premix raw material obtained in step (3) and 1 part of butter are mixed to obtain a mixed dough, which is subjected to secondary low-speed stirring at 130 rpm for 2 min, secondary high-speed stirring at 200 rpm for 3 min, and then allowed to stand and ferment at 25°C until the volume of the mixed dough doubles, to obtain a pre-fermented dough; (5) The pre-fermented dough obtained in step (4) is subjected to secondary fermentation at 35°C until the volume of the pre-fermented dough reaches 1.5 times, and then divided to obtain a bread dough; (6) The bread dough obtained in step (5) is baked at an upper fire temperature of 180°C and a lower fire temperature of 170°C for 20-30 min, to obtain the natural high-folate bread.

[0025] Example 3 A method for increasing the folate content in bread, the method specifically comprises the following steps: (1) Black quinoa seeds are washed, then wetted and soaked, and germinated in the dark for 42 h at a temperature of 25°C and a relative humidity of 90%; after germination, moisture removal is performed at 55°C, followed by drying at 55°C until the moisture content is <10 wt%, and the product is pulverized and sieved through a 100-mesh sieve to obtain germinated quinoa flour.

[0026] (2) In parts by weight, 12 parts of the germinated quinoa flour obtained in step (1), 2 parts of Lactobacillus plantarum powder, 0.05 part of rice bran aliphatic alcohols, 25 parts of water and 12 parts of high-gluten wheat flour are mixed, and the mixture is allowed to stand and culture at 32°C for 20 h to obtain a sourdough; the viable count of the Lactobacillus plantarum powder is 7×10 7 CFU / g.

[0027] (3) In parts by weight, 30 parts of the sourdough obtained in step (2), 40 parts of high-gluten flour, 1 part of active dry yeast, 2 parts of sedoheptulose, 0.6 part of table salt and 30 parts of water are mixed, and the mixture is stirred at 140 rpm at low speed for 3 min to form a dough, and then stirred at 220 rpm at high speed for 4 min to obtain a premix raw material; (4) In parts by weight, 50 parts of the premix raw material obtained in step (3) and 2 parts of butter are mixed to obtain a mixed dough, which is subjected to secondary low-speed stirring at 140 rpm for 3 min, secondary high-speed stirring at 220 rpm for 4 min, and then allowed to stand and ferment at 30°C until the volume of the mixed dough doubles, to obtain a pre-fermented dough; (5) The pre-fermented dough obtained in step (4) is subjected to secondary fermentation at 38°C until the volume of the pre-fermented dough reaches 1.5 times, and then divided to obtain a bread dough; (6) The bread dough obtained in step (5) is baked at an upper fire temperature of 190°C and a lower fire temperature of 180°C for 30 min, to obtain the natural high-folate bread.

[0028] Comparative Example 1 The difference between Comparative Example 1 and Example 1 is that the rice bran fatty alkyl alcohol in step (2) is omitted, while the rest are the same as in Example 1.

[0029] Comparative Example 2 The difference between Comparative Example 2 and Example 1 is that the sedoheptulose in step (3) is replaced with sucrose, while all other components are the same as in Example 1.

[0030] Experimental Example 1 Effects of rice bran fatty alkyl alcohols on the viable count of Lactobacillus plantarum during sourdough fermentation Sourdough prepared in step (2) of Example 1 and Comparative Example 1 were used as test samples. During the static culture of the sourdough, samples were taken at 0 h, 4 h, 8 h, 12 h, and 16 h. At each time point, 10.00 g of sourdough sample was accurately weighed and placed in a homogenizing bag containing 90 mL of sterile physiological saline. The mixture was vortexed until homogeneous, then diluted sequentially to the desired gradient. 100 μL of the diluted solution was spread onto MRS solid medium plates. The plates were incubated at 37 ℃ for 48 h before colony counting. Three replicates were set up for each time point. Colony counts were expressed as log CFU / g sourdough. Results are as follows: Figure 1 As shown.

[0031] The results are as follows Figure 1 The figure shows the effect of fatty alkyl alcohols in rice bran on the viable count of *Lactobacillus plantarum* during the fermentation of sourdough. Figure 1 It was found that the number of viable Lactobacillus plantarum bacteria in the two groups of sourdough were basically the same in the initial stage of fermentation. As the static culture time increased, the number of viable bacteria in Example 1 was significantly higher than that in Comparative Example 1 at all time points. The above results indicate that rice bran fatty alkanols can effectively promote the growth and metabolism of Lactobacillus plantarum in sourdough, significantly increase its biomass, and provide a sufficient cell basis for the biosynthesis of folic acid in the subsequent bread fermentation process.

[0032] Experimental Example 2 Effect of rice bran fatty alkyl alcohols on the 5-methyltetrahydrofolate content in bread To verify the effect of rice bran fatty alkanols on increasing the natural folic acid content in the final bread product, breads baked in step (6) of Example 1, Comparative Example 1, and Comparative Example 2 were used as test samples. The bread samples were freeze-dried and pulverized. 0.5000 g of freeze-dried bread powder was accurately weighed and mixed evenly with 4 mL of potassium phosphate buffer (0.05 mol / L, pH 6.5, containing 1% L-ascorbic acid and 0.1% DTT). The mixture was boiled in a water bath for 5 min, and then rapidly cooled in an ice bath for 5 min. 800 μL of α-amylase (11.84 U / mL) was added, shaken thoroughly, and incubated at 37 ℃ for 30 min. 150 μL of papain (8000 U / mL) was added, shaken to mix, and incubated at 37 ℃ for 1 h. After incubation, the enzyme activity was inactivated by boiling in a water bath for 5 min, and then rapidly cooled in an ice bath for 5 min. The mixture was centrifuged at 4 ℃ and 12000 rpm for 15 min, and the supernatant was collected. Add 1 mL of supernatant to 40 μL of rat serum, shake thoroughly to mix, incubate at 37 ℃ for 4 h, boil in water for 5 min, cool in an ice bath for 5 min, and centrifuge at 4 ℃ and 12000 rpm for 15 min. The entire extraction process was carried out under dark conditions. The supernatant was filtered through a 0.45 μm organic filter membrane, and the content of 5-methyltetrahydrofolate was determined by high performance liquid chromatography-fluorescence detection. Each sample was measured in triplicate, and the results are expressed as mean ± standard deviation, in μg / 100 g bread. The results are shown in Table 1.

[0033] Table 1. Effects of rice bran fatty alkyl alcohols on the 5-methyltetrahydrofolate content in bread. The results are shown in Table 1, illustrating the effect of rice bran fatty alkanols on the 5-methyltetrahydrofolate content in bread. Table 2 shows that, compared to Comparative Example 1, the 5-methyltetrahydrofolate content in the breads of Example 1 and Comparative Example 2 was significantly increased. These results indicate that rice bran fatty alkanols significantly promote the proliferation of *Lactobacillus plantarum* in sourdough, thereby enhancing its ability to biosynthesize folic acid. The combination of *Lactobacillus plantarum* and quinoa seeds significantly increases the folic acid content in the final bread, effectively supplementing the body's folic acid levels. This demonstrates good nutritional value and functionality, is environmentally friendly, and is easy to industrialize. Furthermore, there was no significant difference in folic acid content between Comparative Example 2 and Example 1, indicating that sedulose substitution for sucrose has no negative impact on folic acid synthesis, further confirming that rice bran fatty alkanols are the core functional factor for enhancing folic acid levels.

[0034] Experimental Example 3 Bread quality testing To verify the quality of bread obtained by different preparation methods, the finished bread products obtained in step (6) of Examples 1-3, Comparative Example 1, and Comparative Example 2 were taken as test samples. The specific experimental steps are as follows: (1) After cooling the finished bread to room temperature, weigh it (recorded as m, unit g), and determine the bread volume (recorded as v, unit mL) using the rapeseed displacement method. Bread specific volume = v / m, unit mL / g. Three replicates were performed for each sample, and the average value was taken.

[0035] (2) Cut the finished bread into 40 mm thick slices and select the center of the bread for testing. Use an aluminum cylindrical probe TA-AACC36, test speed 1.00 mm / s, deformation percentage 50%, trigger point load 5g, cycle twice, and measure each sample three times in parallel. Record the hardness, elasticity and chewiness of the bread, and take the average value of the results.

[0036] (3) Twenty trained evaluators (10 men and 10 women, aged 20-40) will score the bread’s appearance, color, texture, flavor and texture according to the sensory evaluation standards in GB / T 20981-2021 General Rules for Bread Quality. The total score is 100 points, and the average score will be taken.

[0037] Table 2. Bread quality indicators for different treatment groups The results are shown in Table 2, which presents the bread quality indicators for different treatment groups. As can be seen from Table 2, compared with Comparative Example 2, the bread prepared in Examples 1-3 of this invention showed a significantly increased specific volume, significantly reduced hardness and chewiness, improved elasticity, and a significantly higher overall sensory score. These results indicate that sedetate-heptanose, as a substitute for sucrose, can significantly improve the specific volume, textural properties, and overall sensory quality of bread, providing consumers with sugar control needs with a new choice of bread products that combine good taste and health benefits.

[0038] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. The basic principles and main features of the present invention have been described above with specific implementation schemes. Based on the present invention, some modifications or substitutions can be made, but these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of protection claimed by the present invention.

Claims

1. A method for increasing the folic acid content in bread, characterized in that, The method specifically includes the following steps: (1) After washing the black quinoa seeds, soak them in water and germinate them in the dark; after germination, dry them, crush them and sieve them to obtain germinated quinoa powder; (2) By weight, take 8-12 parts of sprouted quinoa powder, 1-2 parts of Lactobacillus plantarum powder, 0.01-0.03 parts of rice bran fatty alkyl alcohol, 24-30 parts of water and 14-16 parts of high gluten wheat flour obtained in step (1), mix them, and let them stand to cultivate to obtain sour dough; (3) By weight, take 25-30 parts of sourdough, 35-40 parts of high-gluten flour, 0.6-1 parts of active dry yeast, 1-2 parts of sedum heptose, 0.4-0.6 parts of salt and 25-30 parts of water obtained in step (2), mix them, stir at low speed to form a dough, and then stir at high speed to obtain premixed ingredients. (4) By weight, take 45-50 parts of premixed ingredients obtained in step (3) and 1-2 parts of butter to make a mixed dough. Mix at low speed twice, mix at high speed twice, and let it stand to ferment to obtain the first fermented dough. (5) Take the first fermented dough obtained in step (4), ferment it a second time, divide it, and obtain bread dough; (6) Take the bread dough obtained in step (5) and bake it to obtain natural high folic acid bread.

2. The method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (1), the germination temperature in the dark is 20-25℃, the relative humidity is 85%-90%, and the time is 32-42 h; the drying temperature is 45-55℃; the drying is carried out until the moisture content is <10wt%, and the sieve mesh is 80-100 mesh.

3. The method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (2), the static culture temperature is 28-32℃, and the static culture time is 16-20 h; the viable count of the *Lactobacillus plantarum* powder is 5-7 × 10⁻⁶. 7 CFU / g.

4. The method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (3), the low-speed stirring time is 2-3 minutes and the low-speed stirring speed is 130-140 rpm; the high-speed stirring time is 3-4 minutes and the high-speed stirring speed is 200-220 rpm.

5. The method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (4), the dough is left to ferment until it doubles in volume. The fermentation temperature is 25-30°C. The low-speed mixing time is 2-3 minutes and the low-speed mixing speed is 130-140 rpm. The high-speed mixing time is 3-4 minutes and the high-speed mixing speed is 200-220 rpm.

6. The method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (5), the secondary fermentation is carried out to 1.5 times the volume of the initial fermented dough, and the temperature of the secondary fermentation is 35-38℃.

7. A method for increasing the folic acid content in bread according to claim 1, characterized in that, In step (6), the upper heat temperature is 180–190℃ and the lower heat temperature is 170–180℃; the baking time is 20–30 min.

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