Lactobacillus mucosae Z-15 and its application in the preparation of fermented bread

Abstract

The present invention discloses Lactobacillus mucosae Z-15 and its application in the preparation of fermented bread, belonging to the technical field of microbiology. There is provided Lactobacillus mucosae ( Limosilactobacillus fermentum ) Z-15, which was deposited at the China Center for Type Culture Collection on January 5, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024024. The method for preparing fermented bread includes: mixing Lactobacillus mucosae Z-15, sterile water and high-gluten flour, and standing to obtain a sourdough; mixing with high-gluten flour, Kluyveromyces marxianus GY-8, granulated sugar, salt and water, then adding butter, standing for fermentation, and performing secondary fermentation to obtain bread dough, and baking to obtain fermented bread. The flavor of the bread obtained by co-fermentation of Lactobacillus mucosae Z-15 and Kluyveromyces marxianus GY-8 in the present invention is significantly improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbiology, and particularly relates to Lactobacillus mucosae Z-15 and its application in the preparation of fermented bread. Background Art

[0002] The advantages of sourdough technology in improving bread quality and the characteristics of "green label" have changed the situation where baker's yeast dominates the baking industry. Moreover, sourdough fermentation technology has advantages such as being traditional, natural, and sustainable, and has broad application prospects in the food industry. During the fermentation process of sourdough, lactic acid bacteria ( lactic acid bacteria ) will produce a series of metabolites such as exopolysaccharides and organic acids, and these metabolites have an impact on the rheological properties of the dough, the texture, aging, and flavor of the bread. Acetic acid in the metabolites is an important flavor compound enhancer in bread and can also act as a catalyst for the Maillard reaction together with lactic acid. The acidic environment created by the organic acids activates the activity of endogenous proteases in the fermentation substrate, and under the combined action of the endogenous peptidases contained in the lactic acid bacteria themselves, proteins are decomposed into peptides with smaller molecular weights and free amino acids, thus greatly increasing the content of free amino acids in the bread. Some lactic acid bacteria in sourdough can also synthesize exopolysaccharides in situ, and some of the exopolysaccharides have good hydrophilic properties similar to the hydrocolloids in fermented pasta. Therefore, this part of the exopolysaccharides has the function of replacing hydrocolloids and hindering the migration of water, so it can effectively enhance the anti-aging ability of the product during storage.

[0003] Kluyveromyces marxianus ( Kluyveromyces marxianusIt belongs to the genus Kluyveromyces of the family Saccharomycetaceae and is widely present in natural environments such as koumiss, milk, and fruits. Kluyveromyces marxianus is a food safety-grade yeast, and its safety in the food and medical industries has been confirmed by the National Health Commission of China, the European Food Safety Authority, and the US Food and Drug Administration, meaning it can be consumed by humans. As a new type of yeast, Kluyveromyces marxianus has characteristics such as fast growth rate, high temperature resistance, and high biomass, giving it unique advantages compared to Saccharomyces cerevisiae in production preparation and applications. Currently, Saccharomyces cerevisiae is the preferred baker's yeast in the baking industry. During the alcohol fermentation process, pyruvate decarboxylates to produce carbon dioxide, and the accumulation of carbon dioxide as a by-product makes the bread fluffy, which is a necessary characteristic for baking. In addition, Kluyveromyces marxianus also has a fermentation ability that is not affected by the presence of O2, indicating that Kluyveromyces marxianus has great potential for dough fermentation. Existing commercial yeast powders on the market have characteristics such as fast fermentation speed and stable product quality, but the varieties of fermentation products of commercial yeast powders are few, and the bread produced using commercial yeast has a relatively weak taste, insufficiently plump texture, and the bread is prone to staling. Therefore, there is still a need in the current market for a baker's yeast that has a rich fermentation fragrance and can extend the shelf life of bread to solve the problem of the single fragrance of bread fermented by current baker's yeasts. Summary of the Invention

[0004] To solve the above technical problems, the present invention provides Lactobacillus fermentum Z-15 and its application in the preparation of fermented bread. Lactobacillus fermentum Z-15 is taxonomically named Lactobacillus fermentum Z-15 Limosilactobacillus fermentum Z-15 has the application potential as a sourdough starter. When co-fermented with Kluyveromyces marxianus GY-8, it overcomes the problem of generally low fermentation cell concentration. The maximum value of its fermented dough is comparable to that of commercial yeast, and the flavor of the bread prepared by fermentation is significantly improved.

[0005] To achieve the above object, the present invention provides a Lactobacillus fermentum ( Limosilactobacillus fermentum ) Z-15. The Lactobacillus fermentum Z-15 was deposited at the China Center for Type Culture Collection on January 5, 2024. The deposit address is Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024024.

[0006] The present invention also provides a bacterial agent for preparing fermented bread, including Lactobacillus fermentum Z-15 and Kluyveromyces marxianus GY-8. The Lactobacillus fermentum Z-15 is taxonomically named Lactobacillus fermentum Z-15 Limosilactobacillus fermentum Z-15, with the deposit number CCTCC NO: M2024024, and the Kluyveromyces marxianus GY-8 is taxonomically named Kluyveromyces marxianus GY-8Kluyveromyces marxianus GY-8, with the deposit number of CCTCC NO: M2024025.

[0007] The present invention also provides the application of the fermented Lactobacillus mucosae Z-15 in the preparation of fermented bread.

[0008] The present invention also provides a method for preparing fermented bread using the fermented Lactobacillus mucosae Z-15, comprising the following steps:

[0009] (1) Mix the fermented Lactobacillus mucosae Z-15, sterile water and high-gluten flour, and let stand for cultivation to obtain a sourdough.

[0010] (2) Mix high-gluten flour, the sourdough obtained in step (1), Kluyveromyces marxianus GY-8, granulated sugar, salt and water, initially stir at a low speed to form a dough, and then stir at a high speed to obtain a premixed raw material.

[0011] (3) Mix the premixed raw material obtained in step (2) with butter, stir at a low speed for the second time, stir at a high speed for the second time, and let stand for fermentation until it doubles in volume to obtain a primary fermented dough.

[0012] (4) Ferment the primary fermented dough obtained in step (3) until it reaches 1.5 times the volume of the primary fermented dough, and divide it to obtain bread dough.

[0013] (5) Bake the bread dough obtained in step (4) to obtain fermented bread.

[0014] Preferably, in step (1), the mass ratio of the fermented Lactobacillus mucosae Z-15, sterile water and high-gluten flour is 1:1:1. After mixing the fermented Lactobacillus mucosae Z-15, sterile water and high-gluten flour in step (1), the initial effective viable count of the fermented Lactobacillus mucosae Z-15 is 5×10 7 CFU / g.

[0015] Preferably, in step (1), the temperature for the standing cultivation is 28~32 °C, and the time for the standing fermentation is 8~16 h.

[0016] Preferably, in step (2), the mass ratio of the high-gluten flour, the sourdough obtained in step (1), Kluyveromyces marxianus GY-8, granulated sugar, salt and water is 50~150:15~45:2~6:3~9:0.75~2.25:20~60; the rotation speed for the initial low-speed stirring in step (2) is 140~160 rpm, and the time for the initial low-speed stirring is 2~4 min; the rotation speed for the initial high-speed stirring in step (2) is 200~220 rpm, and the time for the initial high-speed stirring is 2~4 min.

[0017] Preferably, the amount of butter used in step (3) is calculated according to the mass ratio of the sourdough to butter in step (2) of 50-150:4-12; the rotation speed of the secondary low-speed stirring in step (3) is 140-160 rpm, and the time of the secondary low-speed stirring is 1-3 min; the rotation speed of the secondary high-speed stirring in step (3) is 200-220 pm, and the time of the secondary high-speed stirring is 2-4 min; the temperature of the static fermentation in step (3) is 15-25 °C; the temperature of the secondary fermentation in step (4) is 35-39 °C.

[0018] Preferably, the upper heating temperature of the baking in step (4) is 180-190 °C, the lower heating temperature of the baking is 170-180 °C, and the baking time is 20-26 min.

[0019] The present invention also provides the fermented bread prepared by the method for preparing the fermented bread.

[0020] The present invention also provides the application of the Kluyveromyces marxianus GY-8 in the preparation of fermented bread, and the Kluyveromyces marxianus GY-8 is taxonomically named Kluyveromyces marxianus GY-8 Kluyveromyces marxianus GY-8, which was deposited at the China Center for Type Culture Collection on January 5, 2024, with the deposit address of Wuhan University, Wuhan, China, and the deposit number of CCTCC NO: M2024025.

[0021] Compared with the prior art, the present invention has the following advantages and technical effects:

[0022] The Lactobacillus mucosae Z-15 provided by the present invention has the application potential of a sourdough starter. The Kluyveromyces marxianus GY-8 grows rapidly and has the application potential of a main starter, overcoming the problem of generally low fermentation cell concentration. As the fermentation time prolongs, the dough fermentation power gradually increases. When the fermentation time is too long, more than 80 min, the dough is over-proofed and the fermentation power begins to decline. The dough fermented by commercial yeast reaches its maximum fermentation power at 60 min. When the fermentation enters the middle and late stages, the increase in the addition amount has no obvious effect on the fermentation power of Kluyveromyces marxianus GY-8. When the addition amount is 3 g / 100 g flour by wet weight, in the early stage of fermentation, its fermentation power is slightly weaker than that of commercial yeast, while in the late stage of fermentation, the fermentation power of its fermented dough is basically the same as that of the dough fermented by commercial yeast. When the addition amount reaches 4 g / 100 g or more, the maximum value of its fermented dough is the same as that of commercial yeast, and there is also basically no difference after 80 min.

[0023] The co-fermentation of Lactobacillus mucosae Z-15 provided by the present invention and Kluyveromyces marxianus GY-8 can reduce the pH of bread dough and increase the titratable acidity; the specific volume of the prepared fermented bread increases by 3.94%, and the height-diameter ratio decreases; the texture of the bread is improved. The addition of the sourdough prepared from Lactobacillus mucosae Z-15 improves the hardness, chewiness and adhesiveness of the bread, making the bread have a soft and easy-to-chew taste; the addition of the sourdough prepared from Lactobacillus mucosae Z-15 increases the number of pores in the bread and decreases the average pore size, improving the internal structure of the bread, improving the cross-linking degree of the gluten protein network in the dough, and enhancing the stability of the gas pores in the dough. The preparation method of the present invention improves the protein digestibility of the fermented bread in the gastric and intestinal stages, also improves the protein efficiency ratio and amino acid score, and significantly increases the nutritional value of the protein; promotes the accumulation of resistant starch in the fermented bread; reduces the retrogradation enthalpy value of amylopectin during storage. It may be that Lactobacillus mucosae Z-15 produces substances such as organic acids and exopolysaccharides during fermentation, which have inhibitory effects on the recrystallization of amylopectin, and the acidic environment it creates will stimulate the activity of amylase, increase the hydrolysis of amylopectin by amylase, change the crystalline region of starch, and thus affect the aging of starch during storage; extends the storage period of the bread; significantly improves the flavor of the bread, increases the sour taste and aftertaste compared with commercial yeast bread, reduces the astringency and bitterness, improves the taste of the product, and is superior to commercial yeast-fermented bread in terms of texture structure, flavor and texture, with a softer texture, richer taste and higher sensory score. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0025] Figure 1 For the determination of the colony count in the sourdough prepared in Example 1 and the influence of different addition amounts of Kluyveromyces marxianus GY-8 on the dough fermentation power. Among them, A is the determination of the colony count in the sourdough prepared in Example 1, B is the influence of different addition amounts of Kluyveromyces marxianus GY-8 on the dough fermentation power, and SY represents commercial yeast powder;

[0026] Figure 2 For the determination of the pH, TTA, bread specific volume, height-diameter ratio and baking loss of the fermented bread prepared in Example 1 and Comparative Examples 1-2. Among them, A is the determination of pH and TTA, B is the determination of bread specific volume, height-diameter ratio and baking loss, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1;

[0027] Figure 3 For the determination of the bread core capsule structure of the fermented breads prepared in Example 1 and Comparative Examples 1-2, where A is the fermented bread prepared in Comparative Example 2, B is the fermented bread prepared in Comparative Example 1, C is the fermented bread prepared in Example 1, D is the black and white diagram of the fermented bread prepared in Comparative Example 2, E is the black and white diagram of the fermented bread prepared in Comparative Example 1, and F is the black and white diagram of the fermented bread prepared in Example 1;

[0028] Figure 4 For the in vitro protein digestibility of the fermented breads prepared in Example 1 and Comparative Examples 1-2, where SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1;

[0029] Figure 5 For the determination of the retrogradation enthalpy value of the bread amylopectin and the bread core hardness during storage of the fermented breads prepared in Example 1 and Comparative Examples 1-2, where A is the retrogradation enthalpy value of the bread amylopectin, B is the bread core hardness, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1;

[0030] Figure 6 For the determination of the moisture content of each part of the bread during storage of the fermented breads prepared in Example 1 and Comparative Examples 1-2, where A is the moisture content of the bread crust, B is the moisture content at 1 cm from the bread crust, C is the moisture content of the bread core, D is the water activity, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1;

[0031] Figure 7 For the flavor determination of the fermented breads prepared in Example 1 and Comparative Examples 1-2, where A is the electronic nose detection, B is the electronic tongue detection, C is the sensory evaluation, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1. Detailed implementation manners

[0032] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0033] It should be understood that the terms described in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the range.

[0034] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of this invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0035] Without departing from the scope or spirit of this invention, various modifications and variations can be made to the specific embodiments of the specification of this invention, which will be apparent to those skilled in the art. Other embodiments obtained from the specification of this invention will be apparent to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0036] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0037] YPD agar medium and MRS agar medium were purchased from Qingdao Haibo Biotechnology Co., Ltd.; commercial yeast powder was purchased from Angel Yeast Co., Ltd.; BCA kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd.

[0038] Example 1

[0039] Take out GY-8 preserved in a glycerol tube, streak it onto YPD agar medium with an inoculation loop, and perform activation culture at 30 °C and 180 rpm for 18 h; pick a single colony into 10 mL of YPD liquid medium, and culture it at 30 °C and 180 rpm for 12 h for secondary activation; inoculate the secondary-activated GY-8 solution into 100 mL of YPD medium at a ratio of 2% (v / v), culture it until the mid-late logarithmic growth phase, take it out and aliquot it into 50 mL test tubes, centrifuge at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Kluyveromyces marxianus GY-8.

[0040] Take out Z-15 preserved in a glycerol tube, streak it onto MRS agar medium with an inoculation loop, and perform static culture at 37 °C for 24 h; pick a single colony into 10 mL of MRS liquid medium, and perform static culture at 37 °C for 12 h for secondary activation; inoculate the secondary-activated Z-15 solution into 100 mL of MRS liquid medium at a ratio of 2% (v / v), culture it until the mid-late logarithmic growth phase, take it out and aliquot it into 50 mL test tubes, centrifuge at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Lactobacillus fermentum Z-15.

[0041] (1) Lactobacillus mucosae Z-15 was mixed with sterile water, and then mixed with high-gluten flour. It was statically cultured at 30 °C for 12 h to obtain a sourdough; the mass ratio of Lactobacillus mucosae, sterile water and high-gluten flour was 1:1:1. After mixing Lactobacillus mucosae, sterile water and high-gluten flour, the initial effective viable count of Lactobacillus mucosae was 5×10 7 CFU / g.

[0042] (2) 100 g of high-gluten flour, 30 g of sourdough, 4 g of Kluyveromyces marxianus GY-8, 6 g of granulated sugar, 1.5 g of table salt and 40 g of water were mixed. It was initially stirred at a low speed of 150 rpm for 3 min to form a dough, and then initially stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material;

[0043] (3) The premixed raw material was mixed with 8 g of butter. It was stirred at a second low speed of 150 rpm for 2 min, and stirred at a second high speed of 210 rpm for 3 min to extend the gluten. It was rounded and shaped, and statically fermented at 20 °C until it doubled in volume to obtain a primary fermented dough;

[0044] (4) The primary fermented dough was secondarily fermented at 37 °C until it reached 1.5 times the volume of the primary fermented dough. It was divided into portions of 60 g each, and the surface was smoothed to obtain bread dough;

[0045] (5) The bread dough was placed in an oven preheated for 20 min, and baked at an upper fire temperature of 185 °C and a lower fire temperature of 175 °C for 23 min to obtain fermented bread.

[0046] Example 2

[0047] The preserved GY-8 in the glycerol tube was taken out, streaked onto the YPD agar medium with an inoculation loop, and activated and cultured at 30 °C and 180 rpm for 18 h; a single colony was picked into 10 mL of YPD liquid medium and cultured at 30 °C and 180 rpm for 12 h for secondary activation; the secondarily activated GY-8 solution was inoculated into 100 mL of YPD medium at a ratio of 2% (v / v), cultured until the middle and late logarithmic growth phase, taken out and aliquoted into 50 mL test tubes, centrifuged at a low speed of 5000 rpm / min for 5 min, and the cells aggregated at the bottom of the centrifuge tube were collected and washed 3 times repeatedly with distilled water to obtain Kluyveromyces marxianus GY-8.

[0048] Take out the Z-15 preserved in the glycerol tube, streak it onto the MRS agar medium with an inoculation loop, and incubate it statically at 37°C for 24 h; pick a single colony into 10 mL of MRS liquid medium, and incubate it statically at 37°C for 12 h for secondary activation; inoculate the secondary-activated Z-15 solution into 100 mL of MRS liquid medium at a ratio of 2% (v / v), culture it until the mid-late logarithmic growth phase, take it out and aliquot it into 50 mL test tubes, centrifuge it at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Lactobacillus mucosae fermentum Z-15.

[0049] (1) Mix Lactobacillus mucosae fermentum Z-15 with sterile water, and then mix it with high-gluten flour, and incubate it statically at 28°C for 16 h to obtain a sourdough; the mass ratio of Lactobacillus mucosae fermentum, sterile water and high-gluten flour is 1:1:1. After mixing Lactobacillus mucosae fermentum, sterile water and high-gluten flour, the initial effective viable count of Lactobacillus mucosae fermentum is 5×10 7 CFU / g.

[0050] (2) Mix 50 g of high-gluten flour, 15 g of sourdough, 2 g of Kluyveromyces marxianus GY-8, 3 g of granulated sugar, 0.75 g of salt and 20 g of water, stir it at a low speed of 140 rpm for 2 min for the first time to form a dough, and then stir it at a high speed of 200 rpm for 2 min for the first time to obtain a premixed raw material;

[0051] (3) Mix the premixed raw material with 4 g of butter, stir it at a low speed of 140 rpm for 1 min for the second time, and stir it at a high speed of 200 rpm for 2 min for the second time to extend the gluten, roll it into a ball and shape it, and let it ferment statically at 15°C until it doubles in volume to obtain a primary fermented dough;

[0052] (4) Ferment the primary fermented dough at 35°C until it reaches 1.5 times the volume of the primary fermented dough, divide it into portions of 60 g each, and smooth the surface to obtain a bread dough;

[0053] (5) Put the bread dough into an oven preheated for 20 min, set the upper fire temperature to 180°C and the lower fire temperature to 170°C, and bake it for 26 min to obtain a fermented bread.

[0054] Example 3

[0055] Take out GY-8 stored in a glycerol tube, streak it onto YPD agar medium with an inoculation loop, and perform activation culture at 30 °C and 180 rpm for 18 h; pick a single colony into 10 mL of YPD liquid medium, culture it at 30 °C and 180 rpm for 12 h for secondary activation; inoculate the secondary-activated GY-8 solution into 100 mL of YPD medium at a ratio of 2% (v / v), culture it until the middle and late logarithmic growth phase, take it out and aliquot it into 50 mL test tubes, centrifuge it at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Kluyveromyces marxianus GY-8.

[0056] Take out Z-15 stored in a glycerol tube, streak it onto MRS agar medium with an inoculation loop, and perform static culture at 37 °C for 24 h; pick a single colony into 10 mL of MRS liquid medium, and perform static culture at 37 °C for 12 h for secondary activation; inoculate the secondary-activated Z-15 solution into 100 mL of MRS liquid medium at a ratio of 2% (v / v), culture it until the middle and late logarithmic growth phase, take it out and aliquot it into 50 mL test tubes, centrifuge it at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Lactobacillus mucosae fermentum Z-15.

[0057] (1) Mix Lactobacillus mucosae fermentum Z-15 with sterile water, and then mix it with high-gluten flour, and perform static culture at 32 °C for 8 h to obtain sourdough; the mass ratio of Lactobacillus mucosae fermentum, sterile water and high-gluten flour is 1:1:1. After mixing Lactobacillus mucosae fermentum, sterile water and high-gluten flour, the initial effective viable count of Lactobacillus mucosae fermentum is 5×10 7 CFU / g.

[0058] (2) Mix 150 g of high-gluten flour, 45 g of sourdough, 6 g of Kluyveromyces marxianus GY-8, 9 g of granulated sugar, 2.25 g of salt and 60 g of water, stir at a low speed of 160 rpm for the first time for 4 min to form a dough, and then stir at a high speed of 220 rpm for the first time for 4 min to obtain a premixed raw material;

[0059] (3) Mix the premixed raw material with 12 g of butter, stir at a low speed of 160 rpm for the second time for 3 min, and stir at a high speed of 220 rpm for the second time for 4 min to extend the gluten, roll it into a round shape and shape it, and let it stand and ferment at 25 °C until it doubles in volume to obtain a primary fermented dough;

[0060] (4) Ferment the primary fermented dough at 39 °C until it reaches 1.5 times the volume of the primary fermented dough, divide it into portions of 60 g each, and smooth the surface to obtain bread dough;

[0061] (5)Put the bread dough into an oven preheated 20 min in advance, set the upper heating temperature at 190 °C and the lower heating temperature at 170 °C for baking for 20 min to obtain fermented bread.

[0062] Comparative Example 1

[0063] Take out GY-8 preserved in a glycerol tube, streak it onto YPD agar medium with an inoculation loop, and perform activation culture at 30 °C and 180 rpm for 18 h; pick a single colony into 10 mL of YPD liquid medium, culture it at 30 °C and 180 rpm for 12 h for secondary activation; inoculate the secondary-activated GY-8 solution into 100 mL of YPD medium at a ratio of 2% (v / v), culture it until the middle and late logarithmic growth phase, take it out and dispense it into 50 mL test tubes, centrifuge it at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Kluyveromyces marxianus GY-8.

[0064] (1)Mix 100 g of high-gluten flour, 4 g of Kluyveromyces marxianus GY-8, 6 g of granulated sugar, 1.5 g of table salt and 40 g of water, stir at a low speed of 150 rpm for 3 min for the first time to form a dough, and then stir at a high speed of 210 rpm for 3 min for the first time to obtain a premixed raw material;

[0065] (2)Mix the premixed raw material with 8 g of butter, stir at a low speed of 150 rpm for 2 min for the second time, and stir at a high speed of 210 rpm for 3 min for the second time to extend the gluten, roll it into a ball and shape it, and let it stand and ferment at 20 °C until it doubles in volume to obtain a primary fermented dough;

[0066] (3)The primary fermented dough is fermented at 37 °C until it reaches 1.5 times the volume of the primary fermented dough, divided into portions of 60 g each, and the surface is smoothed to obtain bread dough;

[0067] (4)Put the bread dough into an oven preheated 20 min in advance, set the upper heating temperature at 185 °C and the lower heating temperature at 175 °C for baking for 23 min to obtain fermented bread.

[0068] Comparative Example 2

[0069] Take out Z-15 preserved in a glycerol tube, streak it onto MRS agar medium with an inoculation loop, and perform static culture at 37 °C for 24 h; pick a single colony into 10 mL of MRS liquid medium, perform static culture at 37 °C for 12 h for secondary activation; inoculate the secondary-activated Z-15 solution into 100 mL of MRS liquid medium at a ratio of 2% (v / v), culture it until the middle and late logarithmic growth phase, take it out and dispense it into 50 mL test tubes, centrifuge it at a low speed of 5000 rpm / min for 5 min, collect the bacteria aggregated at the bottom of the centrifuge tube, and wash it repeatedly with distilled water 3 times to obtain Lactobacillus mucosae fermentans Z-15.

[0070] (1) Lactobacillus mucosae Z-15 was mixed with sterile water and then with high-gluten flour, and cultured statically at 30 °C for 12 h to obtain a sourdough; the mass ratio of Lactobacillus mucosae, sterile water and high-gluten flour was 1:1:1. After mixing Lactobacillus mucosae, sterile water and high-gluten flour, the initial effective viable count of Lactobacillus mucosae was 5×10⁷ CFU / g.

[0071] (2) 100 g of high-gluten flour, 30 g of sourdough, 4 g of commercial yeast powder, 6 g of granulated sugar, 1.5 g of salt and 40 g of water were mixed, and initially stirred at a low speed of 150 rpm for 3 min to form a dough, and then initially stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material;

[0072] (3) The premixed raw material was mixed with 8 g of butter, stirred at a low speed of 150 rpm for 2 min, and stirred at a high speed of 210 rpm for 3 min to extend the gluten, rounded and shaped, and fermented statically at 20 °C until it doubled in volume to obtain a primary fermented dough;

[0073] (4) The primary fermented dough was secondarily fermented at 37 °C until it reached 1.5 times the volume of the primary fermented dough, divided into portions of 60 g each, and the surface was smoothed to obtain bread dough;

[0074] (5) The bread dough was placed in an oven preheated for 20 min, and baked at an upper fire temperature of 185 °C and a lower fire temperature of 175 °C for 23 min to obtain fermented bread.

[0075] Experimental Example 1

[0076] 1. Determination of the number of colonies during the sourdough fermentation:

[0077] During the fermentation of the sourdough, samples were taken at different time points for a total duration of 36 h; 10 g of sourdough samples were taken at each time point and mixed evenly with 90 mL of sterile physiological saline; diluted to an appropriate concentration by gradient, and 100 μL of the diluted solution was spread on MRS solid medium; the plates after spreading were cultured in a constant temperature incubator at 37 °C for 48 h and then counted. The experiment was set up with 3 replicates, and the results were averaged.

[0078] 2. Determination of the dough fermentation power:

[0079] Fermentation ability is the most important indicator for evaluating the quality of yeast. In this experiment, the measuring cylinder method was used to determine the fermentation ability of yeast dough. Take the activated yeast and centrifuge it at 5000 rpm for 5 min, discard the supernatant, wash the bacterial sludge with sterile normal saline and then centrifuge again, repeat twice; resuspend it in sterile water; make bread dough by mixing flour and water containing yeast in proportion (the experimental operation time is controlled within 5 min), and keep the center temperature of the dough at 27 °C; transfer the dough to a 100 mL long-neck measuring cylinder and mix evenly; ferment it in a constant temperature incubator at 37 °C, and record the volume change every 20 min. The increase in dough height can measure the fermentation ability of yeast. The experiment was set up with 3 replicates, and the results were averaged. The fermentation ability is the ratio of the volume of dough expansion within a specified time to the initial volume of the dough.

[0080] 3. Determination of pH and TTA of bread dough:

[0081] Weigh 10 g of bread dough sample and mix it evenly with 90 mL of distilled water using a magnetic stirrer. After standing for 10 min, measure the pH of the suspension; take 50 mL of the suspension and adjust the pH to 8.5 with 0.10 mol / L NaOH; the volume (mL) of the sodium hydroxide standard solution consumed per milliliter of the suspension is the total acidity. The experiment was set up with 3 replicates, and the results were averaged.

[0082] 4. Determination of bread specific volume, height-to-diameter ratio, and baking loss:

[0083] Bread specific volume: After cooling the made bread to room temperature, weigh it, repeat 3 times and record the results, denoted as m. Measure the volume of the bread, make 3 parallels for each group of samples, and take the average value, denoted as v. Bread specific volume = v / m, and the unit of specific volume is mL / g; the unit of volume v is mL; the unit of mass m is g.

[0084] Height-to-diameter ratio of bread: Measure the height and diameter of the bread with a vernier caliper. The height-to-diameter ratio of bread = bread height / bread diameter.

[0085] Bread baking loss: The baking loss is the ratio of the difference between the weight of the dough and the bread after cooling at room temperature for 2 h to the weight of the dough (fixed at 50 g), and the unit is %. The experiment was set up with 3 replicates, and the results were averaged.

[0086] 5. Analysis of bread texture and internal texture structure:

[0087] Texture detection:

[0088] Cool the bread to room temperature, cut it into bread slices with a thickness of 10 mm, take the two middle slices and stack them for texture measurement, and measure each group of samples 3 times. The measurement parameters of the texture analyzer are: the probe moving rate is 10.0 mm / s, the starting force is 1 N, the compression deformation is set to 50%, the sensing force is 50 N, and the time interval between two compressions is 2 s.

[0089] Internal texture structure analysis:

[0090] Cut the bread into slices with a size of 5×5×2 cm, scan the cut surface of the bread core; use Image J to process the scanned image, and finally obtain the binary pattern image of the bread core and its related parameters. CD represents the number of pores per unit area; TA represents the total pore area; AS represents the average pore size; AF represents the percentage of pore area in the total area.

[0091] 6. Determination of bread protein digestibility and nutritional evaluation:

[0092] (1) Protein digestibility:

[0093] After the prepared bread is freeze-dried, weigh 1.00 g and put it into a digestion tube. Add 15 mL of pepsin solution with a concentration of 20 mg / mL to the digestion tube, and adjust the pH to 1.50. Simulate gastric digestion in a shaker at 37 °C for 3 h; adjust the pH of the digestive juice to 7.00 with 0.20 mol / L NaOH; add 15 mL of trypsin solution with a concentration of 5 mg / mL to the digestion system; simulate intestinal digestion in a shaker at 37 °C for 2 h; add 5 mL of 10% (w / v) trichloroacetic acid solution to inactivate the enzyme. After mixing with a vortex mixer, let it stand at room temperature for 45 min, centrifuge at 10000 rpm for 15 min, take the supernatant, and determine the protein content through a BCA kit. The experiment is set with 3 replicates, and the results are averaged. In vitro protein digestibility (%) = (protein content in the supernatant / protein content in the sample) × 100.

[0094] (2) Protein nutritional evaluation:

[0095] Each test is repeated 3 times and the average value is taken as the test result. First, acid hydrolyze the bread sample, determine the content of hydrolyzed amino acids, and estimate the nutritional indicators of protein components. E / T% is the ratio of the content of 8 essential amino acids to the total amino acids. EAAI represents the essential amino acid index. EAAI is calculated according to the following formula:

[0096] ;

[0097] In the formula, b1, b2... b n is the content of various essential amino acids in the protein to be measured (g / kg); a1, a2... a n is the content of the corresponding essential amino acids in the standard protein (g / kg); n is the number of essential amino acids participating in the calculation.

[0098] BV represents the biological value, which refers to the available part of the test protein and is calculated according to the following formula:

[0099] BV = 1.09×EAAI - 11.7。

[0100] 7. Determination of in vitro digestibility of bread protein and starch:

[0101] The freeze-dried bread samples were successively treated with pepsin, α-amylase, and amyloglucosidase, and then centrifuged to obtain the supernatant. The glucose content was measured using a glucose kit. Among them, rapidly digestible starch (RDS) is the amount of starch hydrolysis within 20 min, slowly digestible starch (SDS) is the amount of starch hydrolysis within 20 - 120 min, and resistant starch refers to the starch content that remains undigested after 120 min of digestion.

[0102] The calculation formulas for rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) are as follows:

[0103] RDS (%) = [((G 20 - G0) × 0.9) / TS] × 100;

[0104] SDS (%) = [((G 120 - G 20 ) × 0.9) / TS] × 100;

[0105] RS (%) = (TS - RDS - SDS) / TS × 100;

[0106] In the above formulas, TS is the total starch content in the bread sample, in mg; G0 is the glucose content in the bread sample before digestion, in mg; G 20 and G 120 refer to the glucose production amounts at 20 min and 120 min of digestion, respectively, in mg.

[0107] 8. Determination of hardness, moisture content, and retrogradation enthalpy of amylopectin during bread storage:

[0108] Hardness:

[0109] The bread was sealed and placed in an incubator at 4°C. Samples were taken at 1, 3, 5, and 7 d of storage, and the hardness of the bread core of the sampled bread was measured. The instrument parameters were set as before. The experiment was set with 3 replicates, and the results were averaged.

[0110] Moisture content:

[0111] Referring to GB5009.3 - 2016, the bread was sealed and packaged and placed in an incubator at 4°C for storage. Samples were taken at 1, 3, 5, and 7 d of storage, and the moisture contents of the bread crust and bread core were measured. The experiment was set with 3 replicates, and the results were averaged.

[0112] Retrogradation enthalpy of amylopectin:

[0113] The bread was sealed and stored in an incubator at 4 °C. Samples of the bread crumb were taken from the bread stored for 1, 3, 5, and 7 days respectively. The sampled samples were freeze-dried, and the enthalpy change was measured using a differential scanning calorimeter. The freeze-dried samples were ground into powder. 2.50 mg of the bread crumb powder and 7.50 μL of distilled water were weighed and added to an aluminum crucible, and then pressed into tablets. They were equilibrated in an incubator at 4 °C for 12 h. The experiment was set up with 3 replicates, and the results were averaged.

[0114] 9. Electronic nose, electronic tongue tests and sensory evaluation of bread

[0115] Electronic nose:

[0116] 3.00 g of bread sample was weighed and placed in a 20 mL sample bottle. By means of headspace collection, the injection needle and the make-up gas needle were inserted into the headspace bottle at the same time, but they were not allowed to touch the sample. The detection conditions of the electronic nose were set as follows: the cleaning time of the sensor was 60 s, and it was repeatedly cleaned until the baseline was zero; the preparation time of the sample was 5 s; the carrier gas was dry and clean air, the carrier gas flow rate was 200 mL / min, and the acquisition time was 120 s.

[0117] Electronic tongue:

[0118] 50.00 g of bread sample was taken and crushed, 150 mL of reference solution was added, homogenized for 10 min, centrifuged at 3000 rpm for 15 min, the supernatant was taken, filtered and then tested with an electronic tongue. 50 mL of the sample solution was poured into a 100 mL sample cup. Before using the electronic tongue, the sensor needs to be activated, that is, the electrodes in it and the sensor need to be immersed in the internal solution and the reference solution for more than 1 day, and a series of calibrations need to be carried out on it before operation to ensure its normality and stability.

[0119] Sensory evaluation:

[0120] Twenty people scored according to the sensory evaluation criteria shown in the following table. The number of men and women was the same, and the age distribution was between 20 and 40 years old. The scoring results were finally averaged. The sensory evaluation criteria refer to the National Standard of the People's Republic of China GB / T 20981-2021 "General Rules for the Quality of Bread".

[0121] 10. Results

[0122] As Figure 1 shown, Lactobacillus mucosae Z-15 grew rapidly and had the application potential as a sourdough starter (as shown in Figure 1 A). The Kluyveromyces marxianus GY-8 provided grew rapidly and had the application potential as a main starter (as shown in Figure 1In B), the problem of generally low fermentation cell concentration is overcome. As the fermentation time extends, the dough fermentation power gradually increases. When the fermentation time is too long, more than 80 min, the dough over-proofs and the fermentation power starts to decline. The dough fermented by commercial yeast reaches its maximum fermentation power at 60 min. When the fermentation enters the middle and late stages, the increase in the addition amount has no obvious effect on the fermentation power of GY-8. When the addition amount is 3 g / 100 g flour by wet weight, in the early stage of fermentation, its fermentation power is slightly weaker than that of commercial yeast, while in the late stage of fermentation, the fermentation power of its fermented dough is basically the same as that of the dough fermented by commercial yeast. When the addition amount reaches more than 4 g / 100 g, the maximum value of its fermented dough is equal to that of commercial yeast, and there is basically no difference after 80 min.

[0123] Table 1 Texture of the fermented breads prepared in Example 1 and Comparative Examples 1-2

[0124] ;

[0125] Note: Different lowercase letters represent significant differences between the data in the same column, P < 0.05.

[0126] Table 2 Structure of the bread cores of the fermented breads prepared in Example 1 and Comparative Examples 1-2

[0127] ;

[0128] Note: AS: Average size of pores; AF: Fraction of pore surface area, i.e., the percentage of the pore area in the total area.

[0129] As Figure 2 shown, the preparation method of co-fermentation of Z-15 and GY-8 provided by the present invention can reduce the pH of the bread dough and increase the titratable acidity (as Figure 2 in A). The specific volume of the bread fermented by Z-15 combined with GY-8 increases by 3.94%, and the height-diameter ratio decreases (as Figure 2 in B). The co-fermentation of Z-15 and GY-8 improves the texture of the bread. The addition of the Z-15 sourdough improves the hardness, chewiness and adhesiveness of the bread, making the bread have a soft and easy-to-chew texture (as shown in Table 1). The addition of the Z-15 sourdough increases the number of pores in the bread and reduces the average size of the pores, improving the internal structure of the bread (as shown in Table 2). As Figure 3 in A, Figure 3 in B, Figure 3 in C, Figure 3 in D, Figure 3 in E and Figure 3 in F shown, the cross-linking degree of the dough gluten protein network is improved, and the stability of the gas pores in the dough is enhanced.

[0130] Table 3 Protein nutritional evaluation of the fermented breads prepared in Example 1 and Comparative Examples 1-2

[0131] ;

[0132] Note: E / T%: the ratio of the content of 8 essential amino acids to the total amino acids; EAAI: essential amino acid index, BV: biological value.

[0133] As Figure 4 shown, the preparation method of the fermented bread of the present invention improves the protein digestibility of the bread in the gastric and intestinal stages, also improves the protein efficiency ratio and amino acid score, and the nutritional value of the protein is significantly increased (as shown in Table 3).

[0134] Table 4 RDS, SDS and RS of the fermented breads prepared in Example 1 and Comparative Examples 1-2

[0135] ;

[0136] Note: RDS: rapidly digestible starch; RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch.

[0137] As shown in Table 4, the preparation method of the fermented bread of the present invention promotes the accumulation of resistant starch in the bread.

[0138] As Figure 5 shown, the preparation method of the fermented bread of the present invention reduces the retrogradation enthalpy value of amylopectin during storage. It may be that the substances such as organic acids and exopolysaccharides produced by strain Z-15 during fermentation have inhibitory effects on the recrystallization of amylopectin, and the acidic environment it creates will stimulate the activity of amylase, increase the hydrolysis of amylopectin by amylase, change the crystalline region of starch, and thus affect the aging of starch during storage (as in Figure 5 A). As in Figure 5 B, during storage, the hardness of the bread crumb of the bread fermented by GY-8 alone is always lower than that of the commercial yeast bread, and the hardness of the bread crumb of the group added with Z-15 sourdough is the lowest. When stored for 7 days, the hardness of the bread fermented by the combination of Z-15 and GY-8 is 10.13 N, and the hardness of the bread fermented by commercial yeast is 14.91 N.

[0139] As Figure 6 shown, the preparation method of the fermented bread of the present invention prolongs the storage period of the bread. As the storage time of the bread prolongs, the moisture content in the bread continuously decreases, and the moisture in the bread crumb migrates to the bread surface. During the migration process, the moisture content at the bread crumb and 1 cm below the bread surface continuously decreases. The water activity of the group fermented by the combination of Z-15 decreases slowly, indicating that the combined fermentation of surface Z-15 sourdough has a certain delaying effect on the aging of the bread, and the bound water of the bread added with Z-15 sourdough is more stable and not easily lost.

[0140] AsFigure 7 As shown, the preparation method of the fermented bread of the present invention significantly improves the flavor of the bread. The response values corresponding to W5S and W1W in the bread after co-fermentation with Z-15 are significantly increased, indicating that it can increase the content of nitrogen oxides and methyl sulfides in the bread; and the overall electronic nose response degree in the bread increases, indicating that the addition of Z-15 sourdough has a great influence on the bread fragrance (such as Figure 7 A in the figure). The sour taste and aftertaste of the co-fermented bread are increased compared with those of the commercial yeast bread, and the astringency and bitterness are reduced, improving the taste of the product (such as Figure 7 B in the figure). The addition of Z-15 sourdough has little influence on the appearance and color of the bread, but is superior to the commercial yeast-fermented bread in terms of texture structure, flavor and texture, with a softer texture, richer taste and higher sensory score (such as Figure 7 C in the figure).

[0141] The above-described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacterial agent composition for preparing fermented bread, characterized in that: It is composed of fermented mucus lactobacillus Z-15 and Kluyveromyces marxianus GY-8, wherein the fermented mucus lactobacillus Z-15 is taxonomically named fermented mucus lactobacillus Z-15 Limosilactobacillus fermentum Z-15, the deposit number is CCTCC NO: M2024024, and the taxonomic name of Kluyveromyces marxianus GY-8 is Kluyveromyces marxianus GY-8 Kluyveromyces marxianus GY-8, the deposit number is CCTCC NO: M2024025.

2. Use of the bacterial agent composition for preparing fermented bread according to claim 1 in preparing fermented bread, characterized in that: The following steps are involved: (1) Fermented Lactobacillus mucilaginosus Z-15, sterile water and high-gluten flour are mixed and cultured to obtain sourdough; (2) mixing high-gluten flour, the sourdough obtained in step (1), Kluyveromyces marxianus GY-8, white sugar, salt and water, stirring at a low speed for the first time to form a dough, and then stirring at a high speed for the first time to obtain a premixed raw material; (3) The premixed raw material obtained in step (2) is mixed with butter, stirred at a low speed for a second time, stirred at a high speed for a second time, and allowed to stand and ferment until the volume is twice the original volume, thereby obtaining a primary fermented dough; (4) The primary fermented dough obtained in step (3) is subjected to secondary fermentation until the volume is 1.5 times of the primary fermented dough, and then divided to obtain bread dough; (5) The bread dough obtained in step (4) is baked to obtain fermented bread.

3. A method for preparing fermented bread using the bacterial agent composition for preparing fermented bread as claimed in claim 1, characterized in that: The following steps are involved: (1) Fermented Lactobacillus mucilaginosus Z-15, sterile water and high-gluten flour are mixed and cultured to obtain sourdough; (2) mixing high-gluten flour, the sourdough obtained in step (1), Kluyveromyces marxianus GY-8, white sugar, salt and water, stirring at a low speed for the first time to form a dough, and then stirring at a high speed for the first time to obtain a premixed raw material; (3) The premixed raw material obtained in step (2) is mixed with butter, stirred at a low speed for a second time, stirred at a high speed for a second time, and allowed to stand and ferment until the volume is twice the original volume, thereby obtaining a primary fermented dough; (4) The primary fermented dough obtained in step (3) is subjected to secondary fermentation until the volume is 1.5 times of the primary fermented dough, and then divided to obtain bread dough; (5) The bread dough obtained in step (4) is baked to obtain fermented bread.

4. The method for preparing fermented bread according to claim 3, characterized in that: The mass ratio of the fermented Lactobacillus mucus Z-15, sterile water and high-gluten flour in step (1) is 1:1:

1. After the fermented Lactobacillus mucus Z-15, sterile water and high-gluten flour are mixed in step (1), the initial effective viable count of the fermented Lactobacillus mucus Z-15 is 5×10 7 CFU / g.

5. The method for preparing fermented bread according to claim 3, characterized in that: The temperature of the static culture in step (1) is 28-32° C., and the time of the static fermentation is 8-16 hours.

6. The method for preparing fermented bread according to claim 3, characterized in that: The mass ratio of the high-gluten flour in step (2), the sour dough obtained in step (1), Kluyveromyces marxianus GY-8, white sugar, salt and water is 50-150:15-45:2-6:3-9:0.75-2.25:20-60; the rotation speed of the initial low-speed stirring in step (2) is 140-160 rpm, and the time of the initial low-speed stirring is 2-4 min; the rotation speed of the initial high-speed stirring in step (2) is 200-220 rpm, and the time of the initial high-speed stirring is 2-4 min.

7. The method for preparing fermented bread according to claim 3, characterized in that: The amount of butter in step (3) is calculated based on the mass ratio of sourdough to butter in step (2) of 50-150:4-12; the rotation speed of the secondary low-speed stirring in step (3) is 140-160 rpm, and the time of the secondary low-speed stirring is 1-3 min; the rotation speed of the secondary high-speed stirring in step (3) is 200-220 pm, and the time of the secondary high-speed stirring is 2-4 min; the temperature of the static fermentation in step (3) is 15-25° C.; the temperature of the secondary fermentation in step (4) is 35-39° C.

8. The method for preparing fermented bread according to claim 3, characterized in that: In step (4), the upper baking temperature is 180-190° C., the lower baking temperature is 170-180° C., and the baking time is 20-26 minutes.

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