Fermented lactobacillus mucus Z-15 and application thereof in preparation of fermented bread

Abstract

The invention discloses fermented lactobacillus mucus Z-15 and application thereof in preparation of fermented bread, belongs to the technical field of microorganisms, and provides the fermented lactobacillus mucus Z-15 which is preserved in the China Center for Type Culture Collection on January 5, 2024, the preservation address is Wuhan University, Wuhan, China, and the preservation number is CCTCC NO: M2024024. The method for preparing the fermented bread comprises the following steps: mixing the fermented lactobacillus mucus Z-15, sterile water and high gluten flour, and standing to obtain sour dough; mixing with high gluten flour, kluyveromyces marxianus GY-8, white granulated sugar, salt and water, then adding butter, standing for fermentation, performing secondary fermentation to obtain bread dough, and baking to obtain the fermented bread. According to the method disclosed by the invention, the flavor of the bread obtained by co-fermentation of the lactobacillus mucus Z-15 and the kluyveromyces marxianus GY-8 is remarkably improved.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and in particular relates to fermented Lactobacillus mucilaginosus Z-15 and application thereof in preparing fermented bread. Background Art

[0002] The sourdough technology has the advantage of improving bread quality and the characteristics of "green label", which has changed the situation that bread yeast dominates the baking industry. In addition, the sourdough fermentation technology has the advantages of 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 extracellular polysaccharides and organic acids, which affect the rheological properties of dough, the texture, aging and flavor of bread. Acetic acid in the metabolites is an important flavor compound enhancer in bread, and can also be used as a catalyst for the Maillard reaction together with lactic acid. The acidic environment created by organic acids activates the activity of endogenous proteases in the fermentation matrix. At the same time, under the joint action of endogenous peptidases contained in lactic acid bacteria themselves, proteins are broken down into peptides with smaller molecular weights and free amino acids, thereby greatly increasing the free amino acid content in bread. Some lactic acid bacteria in sourdough can also synthesize extracellular polysaccharides in situ, and some of the extracellular polysaccharides have good hydrophilic properties similar to those of hydrophilic colloids in fermented pasta. Therefore, this part of the extracellular polysaccharides has the effect of replacing hydrophilic colloids and hindering the migration of water, so it can effectively enhance the anti-aging ability of the product during storage.

[0003] Kluyveromyces marxianus ( Kluyveromyces marxianus) belongs to the genus Kluyveromyces of the family Saccharomyces and is widely found in natural environments such as sour mare's milk, milk, and fruit. Kluyveromyces marxianus is a food-safe yeast. Its safety in the food and medical industries has been confirmed by the National Health Commission of China, the European Food Safety Supervision Agency, and the US Food and Drug Administration, which means it can be eaten by humans. As a new type of yeast, Kluyveromyces marxianus has the characteristics of fast growth, high temperature resistance, and high biomass, which makes it have unique advantages over brewer's yeast in production, preparation, and application. At present, brewer's yeast is the preferred bread yeast in the baking industry. During the alcohol fermentation process, pyruvate is decarboxylated to produce carbon dioxide, which accumulates as a by-product to make the bread fluffy, which is a necessary feature for baking. In addition, Kluyveromyces marxianus also has a fermentation ability that is not affected by the presence of O2, which shows that Kluyveromyces marxianus has great dough fermentation potential. The commercial yeast powder currently available on the market has the characteristics of fast fermentation speed and stable product quality, but the fermentation products of commercial yeast powder are few in variety, and the bread produced using commercial yeast has a relatively light taste, not rich taste, and the bread is easy to age. Therefore, the current market also needs a baker's yeast with rich fermentation aroma and the ability to extend the storage period of bread, so as to solve the problem that the current bread fermented by baker's yeast has a single aroma. Summary of the invention

[0004] In order to solve the above technical problems, the present invention proposes fermented mucus lactobacillus Z-15 and its application in preparing fermented bread. The fermented mucus lactobacillus Z-15 is taxonomically named as fermented mucus lactobacillus Z-15 Limosilactobacillus fermentum Z-15 has the potential for application as a sourdough starter. Co-fermentation with Kluyveromyces marxianus GY-8 overcomes the problem of generally low fermentation bacteria concentration. Its maximum fermentation dough concentration is on par with 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 fermented mucus lactobacillus ( Limosilactobacillus fermentum ) Z-15, the fermented mucus lactobacillus Z-15 was deposited in the China Center for Type Culture Collection on January 5, 2024, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC NO: M2024024.

[0006] The present invention also provides a bacterial agent for preparing fermented bread, comprising 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.

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

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

[0009] Preferably, the mass ratio of the fermented Lactobacillus mucilaginosus Z-15, sterile water and high-gluten flour in step (1) is 1:1:1. After the fermented Lactobacillus mucilaginosus Z-15, sterile water and high-gluten flour are mixed in step (1), the initial effective viable count of the fermented Lactobacillus mucilaginosus Z-15 is 5×10 7 CFU / g.

[0010] Preferably, the temperature of the static culture in step (1) is 28-32° C., and the time of the static fermentation is 8-16 h.

[0011] Preferably, the mass ratio of the high-gluten flour in step (2), the sourdough 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.

[0012] Preferably, the amount of butter in step (3) is calculated according to 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.

[0013] Preferably, the upper baking temperature in step (4) is 180-190° C., the lower baking temperature is 170-180° C., and the baking time is 20-26 minutes.

[0014] The invention also provides the fermented bread prepared by the method for preparing fermented bread.

[0015] The present invention also provides the use of the Kluyveromyces marxianus GY-8 in preparing fermented bread. The taxonomic name of the Kluyveromyces marxianus GY-8 is Kluyveromyces marxianus GY-8 was deposited in the China Center for Type Culture Collection on January 5, 2024, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M2024025.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects: The fermented mucus lactobacillus Z-15 provided by the present invention has the application potential of sour dough starter, and the Kluyveromyces marxianus GY-8 grows rapidly, has the application potential of main starter, and overcomes the problem that the concentration of fermented bacteria is generally low. With the extension of fermentation time, the fermentation power of dough gradually increases. When the fermentation time is too long, greater than 80 minutes, the dough is over-proofed and the fermentation power begins to decrease. The dough fermented by commercial yeast reaches its maximum fermentation power at 60 minutes. 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 3g / 100g of wet weight flour, in the early stage of fermentation, its fermentation power is slightly weaker than that of commercial yeast, and in the late stage of fermentation, the fermentation power of its fermented dough is basically the same as that of commercial yeast fermented dough. When the addition amount reaches more than 4g / 100g, its maximum fermentation dough is equal to that of commercial yeast, and there is basically no difference after 80 minutes.

[0017] The co-fermentation of the fermented Lactobacillus mucilaginosus Z-15 and Kluyveromyces marxianus GY-8 provided by the present invention can reduce the pH of bread dough and improve the titrated acidity; the specific volume of the prepared fermented bread is increased by 3.94%, and the height-to-diameter ratio is reduced; the texture of the bread is improved, and the addition of the sour dough prepared by the fermented Lactobacillus mucilaginosus Z-15 improves the hardness, chewiness and adhesiveness of the bread, so that the bread has a soft and chewy mouthfeel; the addition of the sour dough prepared by the fermented Lactobacillus mucilaginosus Z-15 increases the number of pores in the bread, reduces the average size of the pores, improves the internal structure of the bread, improves the cross-linking degree of the dough gluten protein network, and enhances the stability of the pores in the dough. The preparation method of the present invention improves the protein digestibility of fermented bread in the stomach and intestine stages, and also improves the efficacy ratio and amino acid score of protein, and the nutritional value of protein is significantly increased; the accumulation of resistant starch in fermented bread is promoted; the enthalpy of retrogradation of amylopectin during storage is reduced, which may be due to the fact that organic acids, extracellular polysaccharides and other substances produced by fermented mucus lactobacillus Z-15 during the fermentation process have an inhibitory effect on the recrystallization of amylopectin, and the acidic environment produced by the fermented mucus lactobacillus Z-15 can stimulate the activity of amylase, increase the hydrolysis of amylopectin by amylase, change the crystallization area of ​​starch, and thus affect the aging of starch during storage; the storage period of bread is extended; the flavor of bread is significantly improved, the sourness and aftertaste of commercial yeast bread are increased, the astringency and bitterness are reduced, the product taste is improved, and the texture structure, flavor taste and texture are superior to commercial yeast fermented bread, the texture is softer, the taste is richer, and the sensory score is higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0019] Figure 1 The figures are the determination of the colony count in the sourdough prepared in Example 1 and the effect of different addition amounts of Kluyveromyces marxianus GY-8 on the fermentation ability of the dough, wherein A is the determination of the colony count in the sourdough prepared in Example 1, B is the effect of different addition amounts of Kluyveromyces marxianus GY-8 on the fermentation ability of the dough, and SY represents commercial yeast powder; Figure 2 pH, TTA, bread specific volume, height-to-diameter ratio and baking loss determination of the fermented bread prepared in Example 1 and Comparative Examples 1-2, wherein A is pH and TTA determination, B is bread specific volume, height-to-diameter ratio and baking loss determination, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1; Figure 3The bread core capsule structure of the fermented bread prepared in Example 1 and Comparative Examples 1-2 is measured, wherein 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 picture of the fermented bread prepared in Comparative Example 2, E is the black and white picture of the fermented bread prepared in Comparative Example 1, and F is the black and white picture of the fermented bread prepared in Example 1; Figure 4 The in vitro digestibility of protein of fermented bread prepared in Example 1 and Comparative Examples 1-2, wherein SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1; Figure 5 Determination of the retrogradation enthalpy of amylopectin and the hardness of the bread crumb during storage of the fermented bread prepared in Example 1 and Comparative Examples 1-2, wherein A is the retrogradation enthalpy of amylopectin, B is the hardness of the bread crumb, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1; Figure 6 The moisture content of each part of the fermented bread prepared in Example 1 and Comparative Examples 1-2 during storage was measured, wherein 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; Figure 7 Flavor determination of fermented bread prepared in Example 1 and Comparative Examples 1-2, wherein A is electronic nose detection, B is electronic tongue detection, C is sensory evaluation, SY represents Comparative Example 2, GY-8 represents Comparative Example 1, and GY-8+Z-15 represents Example 1. DETAILED DESCRIPTION

[0020] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0021] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0022] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0023] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to the skilled artisan. The present invention description and examples are exemplary only.

[0024] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0025] 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.; and BCA kit was purchased from Shanghai Bio-Tech Biotechnology Co., Ltd.

[0026] Example 1 The GY-8 stored 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 up and added to 10 mL of YPD liquid medium, and cultured at 30°C and 180 rpm for 12 h for secondary activation; the GY-8 liquid after secondary activation was inoculated into 100 mL of YPD medium at a ratio of 2% (v / v), cultured to the middle and late stages of logarithmic growth, taken out and dispensed into 50 mL test tubes, centrifuged at a low speed of 5000 rpm / min for 5 min, the bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain Kluyveromyces marxianus GY-8.

[0027] The Z-15 stored in the glycerol tube was taken out, streaked onto MRS agar medium with an inoculation loop, and incubated at 37°C for 24 h; a single colony was picked up and added to 10 mL of MRS liquid medium, and incubated at 37°C for 12 h for secondary activation; the Z-15 liquid after secondary activation was inoculated into 100 mL of MRS liquid medium at a ratio of 2% (v / v), and cultured to the middle and late stages of logarithmic growth. The liquid was taken out and dispensed into 50 mL test tubes, and centrifuged at a low speed of 5000 rpm / min for 5 min. The bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain the fermented mucus Lactobacillus Z-15.

[0028] (1) Fermented Lactobacillus mucilaginosus Z-15 was mixed with sterile water and then mixed with high-gluten flour. The mixture was cultured at 30°C for 12 h to obtain sourdough. The mass ratio of fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour was 1:1:1. After the fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour were mixed, the initial effective viable count of the fermented Lactobacillus mucilaginosus was 5×10 7 CFU / g.

[0029] (2) 100 g of high-gluten flour, 30 g of sourdough, 4 g of Kluyveromyces marxianus GY-8, 6 g of white sugar, 1.5 g of salt and 40 g of water were mixed, and the mixture was initially stirred at a low speed of 150 rpm for 3 min to form a mass, and then initially stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material; (3) Mix the premixed raw materials with 8 g of butter, stir at a low speed of 150 rpm for 2 min, and stir at a high speed of 210 rpm for 3 min to stretch the gluten, roll into a round shape, and let it stand at 20°C to ferment until it doubles its original volume to obtain the primary fermented dough; (4) The primary fermented dough is fermented for a second time at 37°C until the volume is 1.5 times of the primary fermented dough, and the dough is divided into portions of 60 g each and the surface is smoothed to obtain bread dough; (5) Put the bread dough into an oven that has been preheated for 20 minutes, set the upper temperature to 185°C and the lower temperature to 175°C, and bake for 23 minutes to obtain fermented bread.

[0030] Example 2 The GY-8 stored 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 up and added to 10 mL of YPD liquid medium, and cultured at 30°C and 180 rpm for 12 h for secondary activation; the GY-8 liquid after secondary activation was inoculated into 100 mL of YPD medium at a ratio of 2% (v / v), cultured to the middle and late stages of logarithmic growth, taken out and dispensed into 50 mL test tubes, centrifuged at a low speed of 5000 rpm / min for 5 min, the bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain Kluyveromyces marxianus GY-8.

[0031] The Z-15 stored in the glycerol tube was taken out, streaked onto MRS agar medium with an inoculation loop, and incubated at 37°C for 24 h; a single colony was picked up and added to 10 mL of MRS liquid medium, and incubated at 37°C for 12 h for secondary activation; the Z-15 liquid after secondary activation was inoculated into 100 mL of MRS liquid medium at a ratio of 2% (v / v), and cultured to the middle and late stages of logarithmic growth. The liquid was taken out and dispensed into 50 mL test tubes, and centrifuged at a low speed of 5000 rpm / min for 5 min. The bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain the fermented mucus Lactobacillus Z-15.

[0032] (1) Fermented Lactobacillus mucilaginosus Z-15 was mixed with sterile water and then mixed with high-gluten flour. The mixture was cultured at 28°C for 16 h to obtain sourdough. The mass ratio of fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour was 1:1:1. After the fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour were mixed, the initial effective viable count of the fermented Lactobacillus mucilaginosus was 5×10 7 CFU / g.

[0033] (2) 50 g of high-gluten flour, 15 g of sourdough, 2 g of Kluyveromyces marxianus GY-8, 3 g of white sugar, 0.75 g of salt and 20 g of water were mixed, stirred at a low speed of 140 rpm for 2 min to form a mass, and then stirred at a high speed of 200 rpm for 2 min to obtain a premixed raw material; (3) Mix the premixed raw materials with 4 g butter, stir at a low speed of 140 rpm for 1 min, and stir at a high speed of 200 rpm for 2 min to stretch the gluten, roll into a round shape, and let it stand at 15°C to ferment until it doubles its original volume to obtain the primary fermented dough; (4) The primary fermented dough is fermented for a second time at 35°C until the volume is 1.5 times of the primary fermented dough, and then divided into portions of 60 g each and the surface is smoothed to obtain bread dough; (5) Put the bread dough into an oven that has been preheated for 20 minutes, set the upper temperature to 180°C and the lower temperature to 170°C, and bake for 26 minutes to obtain fermented bread.

[0034] Example 3 The GY-8 stored 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 up and added to 10 mL of YPD liquid medium, and cultured at 30°C and 180 rpm for 12 h for secondary activation; the GY-8 liquid after secondary activation was inoculated into 100 mL of YPD medium at a ratio of 2% (v / v), cultured to the middle and late stages of logarithmic growth, taken out and dispensed into 50 mL test tubes, centrifuged at a low speed of 5000 rpm / min for 5 min, the bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain Kluyveromyces marxianus GY-8.

[0035] The Z-15 stored in the glycerol tube was taken out, streaked onto MRS agar medium with an inoculation loop, and incubated at 37°C for 24 h; a single colony was picked up and added to 10 mL of MRS liquid medium, and incubated at 37°C for 12 h for secondary activation; the Z-15 liquid after secondary activation was inoculated into 100 mL of MRS liquid medium at a ratio of 2% (v / v), and cultured to the middle and late stages of logarithmic growth. The liquid was taken out and dispensed into 50 mL test tubes, and centrifuged at a low speed of 5000 rpm / min for 5 min. The bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain the fermented mucus Lactobacillus Z-15.

[0036] (1) The fermented Lactobacillus mucilaginosus Z-15 was mixed with sterile water and then mixed with high-gluten flour. The mixture was cultured at 32°C for 8 h to obtain sourdough. The mass ratio of the fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour was 1:1:1. After the fermented Lactobacillus mucilaginosus, sterile water and high-gluten flour were mixed, the initial effective viable count of the fermented Lactobacillus mucilaginosus was 5×10 7 CFU / g.

[0037] (2) 150 g of high-gluten flour, 45 g of sourdough, 6 g of Kluyveromyces marxianus GY-8, 9 g of white sugar, 2.25 g of salt and 60 g of water were mixed, and the mixture was initially stirred at a low speed of 160 rpm for 4 min to form a mass, and then initially stirred at a high speed of 220 rpm for 4 min to obtain a premixed raw material; (3) Mix the premixed raw materials with 12 g of butter, stir at a low speed of 160 rpm for 3 min, and stir at a high speed of 220 rpm for 4 min to stretch the gluten, roll into a round shape, and let it stand at 25°C to ferment until it doubles its original volume to obtain the primary fermented dough; (4) The primary fermented dough is fermented at 39°C for a second time until the volume is 1.5 times of the primary fermented dough, and the dough is divided into portions of 60 g each and the surface is smoothed to obtain bread dough; (5) Put the bread dough into an oven that has been preheated for 20 minutes, set the upper temperature to 190°C and the lower temperature to 170°C, and bake for 20 minutes to obtain fermented bread.

[0038] Comparative Example 1 The GY-8 stored 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 up and added to 10 mL of YPD liquid medium, and cultured at 30°C and 180 rpm for 12 h for secondary activation; the GY-8 liquid after secondary activation was inoculated into 100 mL of YPD medium at a ratio of 2% (v / v), cultured to the middle and late stages of logarithmic growth, taken out and dispensed into 50 mL test tubes, centrifuged at a low speed of 5000 rpm / min for 5 min, the bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain Kluyveromyces marxianus GY-8.

[0039] (1) 100 g high-gluten flour, 4 g Kluyveromyces marxianus GY-8, 6 g white sugar, 1.5 g salt and 40 g water were mixed, stirred at a low speed of 150 rpm for 3 min to form a mass, and then stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material; (2) Mix the premixed raw materials with 8 g of butter, stir at a low speed of 150 rpm for 2 min, and stir at a high speed of 210 rpm for 3 min to stretch the gluten, roll into a round shape, and let it stand at 20°C to ferment until it doubles its original volume to obtain the primary fermented dough; (3) The primary fermented dough is fermented for a second time at 37°C until the volume is 1.5 times of the primary fermented dough, and the dough is divided into portions of 60 g each and the surface is smoothed to obtain bread dough; (4) Put the bread dough into an oven that has been preheated for 20 minutes, set the upper temperature to 185°C and the lower temperature to 175°C, and bake for 23 minutes to obtain fermented bread.

[0040] Comparative Example 2 The Z-15 stored in the glycerol tube was taken out, streaked onto MRS agar medium with an inoculation loop, and incubated at 37°C for 24 h; a single colony was picked up and added to 10 mL of MRS liquid medium, and incubated at 37°C for 12 h for secondary activation; the Z-15 liquid after secondary activation was inoculated into 100 mL of MRS liquid medium at a ratio of 2% (v / v), and cultured to the middle and late stages of logarithmic growth. The liquid was taken out and dispensed into 50 mL test tubes, and centrifuged at a low speed of 5000 rpm / min for 5 min. The bacteria accumulated at the bottom of the centrifuge tube were collected, and washed repeatedly with distilled water for 3 times to obtain the fermented mucus Lactobacillus Z-15.

[0041] (1) Fermented Lactobacillus mucus Z-15 was mixed with sterile water and then mixed with high-gluten flour. The mixture was incubated at 30°C for 12 h to obtain sourdough. The mass ratio of fermented Lactobacillus mucus, sterile water and high-gluten flour was 1:1:1. After the fermented Lactobacillus mucus, sterile water and high-gluten flour were mixed, the initial effective viable count of the fermented Lactobacillus mucus was 5×107 CFU / g.

[0042] (2) 100 g high-gluten flour, 30 g sourdough, 4 g commercial yeast powder, 6 g white sugar, 1.5 g salt and 40 g water were mixed, stirred at a low speed of 150 rpm for 3 min to form a mass, and then stirred at a high speed of 210 rpm for 3 min to obtain a premixed raw material; (3) Mix the premixed raw materials with 8 g of butter, stir at a low speed of 150 rpm for 2 min, and stir at a high speed of 210 rpm for 3 min to stretch the gluten, roll into a round shape, and let it stand at 20°C to ferment until it doubles its original volume to obtain the primary fermented dough; (4) The primary fermented dough is fermented for a second time at 37°C until the volume is 1.5 times of the primary fermented dough, and the dough is divided into portions of 60 g each and the surface is smoothed to obtain bread dough; (5) Put the bread dough into an oven that has been preheated for 20 minutes, set the upper temperature to 185°C and the lower temperature to 175°C, and bake for 23 minutes to obtain fermented bread.

[0043] Experimental Example 1 1. Determination of colony count during sourdough fermentation: During the fermentation process of sourdough, samples were taken at different time points for a total of 36 hours; 10g of sourdough sample was taken at each time point and mixed evenly in 90mL of sterile saline; gradient dilution was performed to an appropriate concentration, and 100μL of the dilution was spread on MRS solid culture medium; the spread plate was incubated in a 37℃ constant temperature incubator for 48 hours and then counted. The experiment was repeated 3 times and the results were averaged.

[0044] 2. Determination of dough fermentation power: Fermentation power is the most important indicator for evaluating yeast quality. This test uses the measuring cylinder method to determine the fermentation power of yeast dough. Take the activated yeast and centrifuge it at 5000rpm for 5 minutes, discard the supernatant, wash the bacterial sludge with sterile saline and centrifuge it twice; resuspend it in sterile water; make bread dough with flour and water containing yeast in proportion (the test operation time is controlled within 5 minutes), and make the center temperature of the dough at 27℃; transfer the dough to a 100mL long-necked measuring cup and mix it evenly; ferment it in a constant temperature incubator at 37℃, and record the volume change every 20 minutes. The increase in dough height can measure the fermentation power of yeast. The experiment was repeated 3 times, and the results were averaged. The fermentation power is the ratio of the volume of the dough expanded within a specified time to the initial volume of the dough.

[0045] 3. Determination of bread dough pH and TTA: Weigh 10g bread dough sample and stir it with 90mL distilled water using a magnetic stirrer. After standing for 10min, measure the pH of the suspension. Take 50mL of the suspension and adjust the pH to 8.5 with 0.10mol / L NaOH. The volume (mL) of sodium hydroxide standard solution consumed per milliliter of suspension is the total acidity. The experiment was repeated 3 times and the results were averaged.

[0046] 4. Determination of bread specific volume, height-to-diameter ratio and baking loss: Bread specific volume: Cool the bread to room temperature and weigh it. Repeat 3 times and record the results, recorded as m. Measure the volume of the bread. Make 3 parallels for each group of samples and take the average value of the results, recorded as v. Bread specific volume = v / m, specific volume unit is mL / g; volume v unit is mL; mass m unit is g.

[0047] Height-to-diameter ratio of bread: Use a vernier caliper to measure the height and diameter of the bread. Height-to-diameter ratio of bread = bread height / bread diameter.

[0048] Baking loss of bread: Baking loss is the ratio of the weight difference between the dough and the bread after cooling at room temperature for 2 hours to the weight of the dough (fixed at 50g), expressed in %. The experiment was repeated 3 times and the results were averaged.

[0049] 5. Analysis of bread texture and internal texture structure: Texture testing: The bread was cooled to room temperature and cut into 10 mm thick slices. The two middle slices were stacked for texture measurement. Each group of samples was measured 3 times. The measurement parameters of the texture analyzer were: probe movement rate 10.0 mm / s, starting force 1 N, compression deformation set to 50%, induction force 50 N, and the time interval between two compressions was 2 s.

[0050] Internal texture structure analysis: The bread was cut into slices of 5×5×2 cm, and the cross section of the bread core was scanned. The scanned image was processed using Image J, and finally the binary pattern of the bread core and its related parameters were obtained. CD represents the number of pores per unit area; TA represents the total area of ​​pores; AS represents the average size of pores; and AF represents the percentage of pore area to total area.

[0051] 6. Determination of bread protein digestibility and nutritional evaluation: (1) Protein digestibility: After freeze-drying, 1.00 g of the prepared bread was weighed and placed in a digestive tube. 15 mL of 20 mg / mL pepsin solution was added to the digestive tube, and the pH was adjusted to 1.50. The digestion was simulated in a shaker at 37 °C for 3 h. The pH of the digestive solution was adjusted to 7.00 with 0.20 mol / L NaOH. 15 mL of 5 mg / mL trypsin solution was added to the digestive system. The digestion was simulated in a shaker at 37 °C for 2 h. 5 mL of 10% (w / v) trichloroacetic acid solution was added to inactivate the enzyme. After mixing with a vortexer, the mixture was allowed to stand at room temperature for 45 min, centrifuged at 10,000 rpm for 15 min, and the supernatant was taken to determine the protein content using a BCA kit. The experiment was repeated 3 times, and the results were averaged. In vitro protein digestibility (%) = (protein content in supernatant / protein content in sample) × 100.

[0052] (2) Protein nutrition evaluation: Each test was repeated 3 times and the average value was taken as the test result. The bread samples were first acid-hydrolyzed to determine the hydrolyzed amino acid content and estimate the nutritional index of the protein component. E / T% is the ratio of the content of 8 essential amino acids to the total amino acids. EAAI stands for the essential amino acid index. EAAI is calculated according to the following formula: ; Where b1, b2...b n is the content of various essential amino acids in the protein to be tested (g / kg); a1, a2...a n is the content of the corresponding essential amino acid in the standard protein (g / kg); n is the number of essential amino acids involved in the calculation.

[0053] BV stands for biological value, which refers to the available fraction of the test protein and is calculated according to the following formula: BV=1.09×EAAI-11.7.

[0054] 7. Determination of in vitro digestibility of bread protein starch: The freeze-dried bread samples were treated with pepsin, α-amylase and amyloglucosidase in sequence and then centrifuged to obtain the supernatant, which was then measured using a glucose kit to obtain the glucose content; rapidly digestible starch (RDS) refers to the amount of starch hydrolyzed within 20 min, slowly digestible starch (SDS) refers to the amount of starch hydrolyzed within 20-120 min, and resistant starch refers to the starch content that has not been digested after 120 min of digestion.

[0055] The calculation formulas for rapidly digestible starch (RDS), slowly digestible starch (SDS), and resistant starch (RS) are as follows: RDS(%)=[(G 20 -G0) × 0.9] / TS × 100; SDS(%)=[(G 120 -G 20 )×0.9] / TS×100; RS(%)=(TS-RDS-SDS) / TS×100; In the above formula, TS is the total starch content in the bread sample, in mg; G0 is the glucose content of the bread sample before digestion, in mg; G 20 , G 120 It refers to the amount of glucose produced after 20 minutes and 120 minutes of digestion respectively, in mg.

[0056] 8. Determination of hardness, moisture and amylopectin recovery enthalpy during bread storage: hardness: The bread was sealed and placed in a 4°C constant temperature box. Samples were taken at 1, 3, 5 and 7 days of storage, and the crumb hardness of the sampled bread was measured. The instrument parameters were set as before. The experiment was repeated 3 times and the results were averaged.

[0057] Moisture Content: Referring to GB5009.3-2016, the bread was sealed and stored in a 4°C constant temperature box. Samples were taken at 1, 3, 5 and 7 days of storage to measure the moisture content of the bread crust and bread core. The experiment was repeated 3 times and the results were averaged.

[0058] Amylopectin recovery enthalpy: The bread was sealed and packaged and stored in a 4°C thermostat. The bread crumbs were sampled after 1, 3, 5, and 7 days of storage, and the sampled samples were freeze-dried. The enthalpy change was measured using a differential scanning calorimeter. The freeze-dried samples were ground into powder, and 2.50 mg of bread crumb powder and 7.50 μL of distilled water were added to an aluminum crucible and pressed into tablets. The samples were balanced in a 4°C thermostat for 12 hours. The experiment was repeated 3 times and the results were averaged.

[0059] 9. Bread electronic nose, electronic tongue test and sensory evaluation Electronic nose: Weigh 3.00g bread sample into a 20mL sample bottle, use the headspace collection method, insert the injection needle and the gas replenishment needle into the headspace bottle at the same time, but do not let them touch the sample. The electronic nose detection conditions are set as follows: the sensor cleaning time is 60s, and the cleaning is repeated until the baseline is zero; the sample preparation time is 5s; the carrier gas is dry and clean air, the carrier gas flow rate is 200mL / min, and the data collection time is 120s.

[0060] Electronic tongue: Take 50.00g bread sample and crush it, add 150mL reference solution, homogenize for 10min, centrifuge at 3000rpm for 15min, take the supernatant, filter it and then test the electronic tongue. Pour 50mL sample solution into a 100mL sample cup. Before using the electronic tongue, the sensor needs to be activated, that is, the electrodes and sensors need to be soaked in the internal solution and reference solution for more than 1d, and a series of calibrations need to be performed before operation to ensure its normality and stability.

[0061] Sensory evaluation: The sensory evaluation criteria are evaluated by 20 people, with an equal number of men and women, aged between 20 and 40 years old, according to the sensory evaluation criteria shown in the table below. The scoring results are finally averaged. The sensory evaluation criteria refer to the National Standard of the People's Republic of China GB / T20981-2021 "General Principles for Bread Quality".

[0062] 10. Results like Figure 1 As shown in Figure 2, the fermentative Lactobacillus mucilaginosus Z-15 grew rapidly and had the potential to be used as a sourdough starter (e.g. Figure 1 The provided Kluyveromyces marxianus GY-8 grows rapidly and has the potential to be used as a main fermentation culture (e.g. Figure 1B), to overcome the problem of generally low fermentation bacteria concentration. As the fermentation time increases, the fermentation power of the dough gradually increases. When the fermentation time is too long, more than 80 minutes, the dough is over-proofed and the fermentation power begins to decline. The dough fermented with commercial yeast reaches its maximum fermentation power at 60 minutes. When the fermentation enters the middle and late stages, the increase in the amount added has no obvious effect on the fermentation power of GY-8. When the addition amount is 3g / 100g flour by wet weight, its fermentation power is slightly weaker than that of commercial yeast in the early stage of fermentation, and in the later stage of fermentation, the fermentation power of its fermented dough is basically the same as that of commercial yeast fermented dough. When the addition amount reaches 4g / 100g or more, its maximum fermentation dough is the same as that of commercial yeast, and there is basically no difference after 80 minutes.

[0063] Table 1 Texture of fermented bread prepared in Example 1 and Comparative Examples 1-2 ;

[0064] Note: Different lowercase letters represent significant differences between data in the same column, P < 0.05.

[0065] Table 2 Crumb structure of fermented bread prepared in Example 1 and Comparative Examples 1-2 ;

[0066] Note: AS: average size of pores; AF: pore surface integral fraction, i.e. the percentage of pore area to total area.

[0067] like Figure 2 As shown, the preparation method of co-fermentation of Z-15 and GY-8 provided by the present invention can reduce the pH of bread dough and increase the titratable acidity (such as Figure 2 The specific volume of Z-15 combined with GY-8 fermented bread increased by 3.94%, and the height-to-diameter ratio decreased (e.g. Figure 2 B). Z-15 combined with GY-8 fermentation improved the texture of bread. The addition of Z-15 sour dough improved the hardness, chewiness and adhesiveness of bread, making the bread soft and chewy (as shown in Table 1). The addition of Z-15 sour dough increased the number of pores in the bread and reduced the average pore size, thus improving the internal structure of the bread (as shown in Table 2). Figure 3 Middle A, Figure 3 Middle B, Figure 3 Middle C, Figure 3 Middle D, Figure 3 ZhongE and Figure 3 As shown in Figure 5(F), the cross-linking degree of the dough gluten protein network was improved and the stability of the pores in the dough was enhanced.

[0068] Table 3 Protein nutritional evaluation of fermented bread prepared in Example 1 and Comparative Examples 1-2 ;

[0069] Note: E / T%: ratio of 8 essential amino acids to total amino acids; EAAI: essential amino acid index, BV: biological value.

[0070] like Figure 4 As shown in Table 3, the method for preparing fermented bread of the present invention improves the protein digestibility of bread in the stomach and intestines, and also improves the efficacy ratio and amino acid score of protein, and the nutritional value of protein is significantly increased (as shown in Table 3).

[0071] Table 4 RDS, SDS and RS of fermented bread prepared in Example 1 and Comparative Examples 1-2 ;

[0072] Note: RDS: rapidly digestible starch; SDS: slowly digestible starch; RS: resistant starch.

[0073] As shown in Table 4, the method for preparing fermented bread of the present invention promotes the accumulation of resistant starch in bread.

[0074] like Figure 5 As shown in the figure, the preparation method of fermented bread of the present invention reduces the enthalpy of retrogradation of amylopectin during storage. It may be that the organic acid, extracellular polysaccharide and other substances produced by the Z-15 strain during the fermentation process have an inhibitory effect on the recrystallization of amylopectin, and the acidic environment created by the strain stimulates the activity of amylase, increases the hydrolysis of amylopectin by amylase, changes the crystallization area of ​​starch, and thus affects the aging of starch during storage (such as Figure 5 (A). Figure 5 Middle B, during storage, the hardness of the bread crumb fermented with GY-8 alone was always lower than that of the commercial yeast bread, and the bread crumb hardness of the sourdough group with Z-15 was the lowest. After 7 days of storage, the hardness of the bread fermented with Z-15 and GY-8 was 10.13N, and the hardness of the bread fermented with commercial yeast was 14.91N.

[0075] like Figure 6 As shown, the preparation method of fermented bread of the present invention prolongs the storage period of bread. As the storage time of bread increases, the moisture content in the bread continues to decrease, and the moisture in the bread core continues to migrate to the bread crust. During the migration process, the moisture content of the bread core and 1 cm below the bread crust continues to decrease. The water activity of the Z-15 co-fermentation group decreases slowly, and the co-fermentation of the surface Z-15 sour dough has a certain delaying effect on the aging of bread, and the bread bound water added with Z-15 sour dough is more stable and not easy to lose.

[0076] like Figure 7As shown in the figure, the preparation method of fermented bread of the present invention significantly improves the flavor of bread. The response values ​​corresponding to W5S and W1W in the bread after Z-15 co-fermentation are significantly increased, indicating that it can increase the content of nitrogen oxides and methyl sulfide in bread; and the overall electronic nose responsiveness in bread increases, indicating that the addition of Z-15 sour dough has a greater impact on the flavor of bread (such as Figure 7 Compared with commercial yeast bread, the sourness and aftertaste of co-fermented bread are increased, the astringency and bitterness are reduced, and the taste of the product is improved (such as Figure 7 The addition of Z-15 sourdough had little effect on the appearance and color of bread, but was superior to commercial yeast-fermented bread in terms of texture, flavor, and texture, with a softer texture, richer taste, and higher sensory scores (e.g. Figure 7 Middle C).

[0077] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A bacterial agent for preparing fermented bread, characterized in that: The invention comprises fermented mucus lactobacillus Z-15 and Kluyveromyces marxianus GY-8, wherein the fermented mucus lactobacillus Z-15 is taxonomically named as 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 fermented Lactobacillus mucilaginosus Z-15 as claimed in claim 1 in preparing fermented bread.

3. A method for preparing fermented bread using the fermented Lactobacillus mucilaginosus Z-15 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.

9. The fermented bread prepared by the method for preparing fermented bread according to any one of claims 3 to 8.

10. The use of Kluyveromyces marxianus GY-8 in preparing fermented bread as claimed in claim 3, characterized in that: The Kluyveromyces marxianus GY-8 is taxonomically named Kluyveromyces marxianus GY-8 Kluyveromyces marxianus GY-8, the deposit number is CCTCC NO: M2024025.

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