A low-sugar bread yeast culture medium and fermentation method capable of replacing molasses

Abstract

This invention belongs to the field of microbial fermentation and bioengineering technology, specifically disclosing a low-sugar baker's yeast culture medium and fermentation method that can replace molasses. The culture medium of this invention does not contain molasses, uses glucose as the main carbon source, and is supplemented with nitrogen and phosphorus sources, potassium, magnesium, and calcium salts, trace elements such as zinc, iron, manganese, and iodine, vitamins such as inositol, vitamin B5, biotin, and para-aminobenzoic acid, and amino acids such as isoleucine, lysine, and histidine. The fermentation method includes seed culture preparation, high-density fermentation culture, and a post-induction treatment 1 hour after the nitrogen and phosphorus sources are depleted by adjusting temperature, pH, rotation speed, and aeration. This invention can obtain low-sugar fresh yeast with high fermentation power and good storage stability suitable for low-sugar dough without using molasses.

Description

Technical Field

[0001] This invention belongs to the field of microbial fermentation and bioengineering technology, and specifically relates to a low-sugar baker's yeast culture medium that can replace molasses, and a method for using the culture medium to carry out high-density fermentation and obtain fresh yeast for low-sugar dough. Background Technology

[0002] Fresh yeast is a commonly used biological leavening agent in the production of fermented dough products such as steamed buns, dumplings, twisted rolls, and bread. Depending on the sugar content of the dough and the application scenario, fresh yeast can generally be divided into low-sugar and high-sugar types. Low-sugar fresh yeast is mainly used in dough systems with no or low sugar content. The leavening power, post-storage leavening power, and fermentation start-up speed of low-sugar fresh yeast directly affect the volume, texture, and production stability of the dough products.

[0003] Currently, most industrial production of fresh yeast uses molasses as the primary carbon source. Besides fermentable sugars such as sucrose, glucose, and fructose, molasses also contains a certain amount of nitrogenous substances, minerals, and B vitamins, thus promoting yeast growth and cell physiological processes to some extent. However, molasses is significantly affected by the sugar-producing season, origin, transportation, storage, and batch composition, resulting in supply and quality fluctuations, as well as difficulties in controlling the production process.

[0004] Glucose has a stable source, well-defined composition, and a clear metabolic pathway, making it suitable as the primary carbon source for constructing a culture system with controllable composition. However, when glucose is used alone to replace molasses, the culture system lacks the auxiliary nutritional factors such as amino acids, vitamins, and trace elements that are naturally present in molasses. This can lead to problems such as low initial fermentation power, rapid decline in fermentation power after storage, and insufficient cellular stress resistance in the resulting low-sugar fresh yeast.

[0005] Therefore, it is necessary to establish a compound culture medium that does not rely on molasses, uses glucose as the main carbon source, and has a relatively well-defined composition. Combined with the induction and regulation process in the later stage of fermentation, this will improve the storage stability of yeast while maintaining its initial fermentation power, thereby meeting the needs of low-sugar fresh yeast production and application. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a low-sugar baker's yeast culture medium that can replace molasses, solving the problems of strong dependence on molasses in the production of low-sugar fresh yeast and insufficient yeast fermentation power and storage stability obtained by glucose-only carbon source culture systems. Another objective of the present invention is to provide a method for high-density fermentation using this culture medium to obtain low-sugar dough fresh yeast.

[0007] This invention is achieved through the following technical solution: A low-sugar baker's yeast culture medium that can replace molasses is a glucose-based compound culture medium without added molasses, using glucose as the main carbon source. The glucose-based compound culture medium consists of a base culture medium and a feed culture medium. The base culture medium uses sterile deionized water as a solvent and contains glucose, magnesium sulfate, zinc sulfate, ferrous sulfate, manganese sulfate, potassium iodide, inositol, vitamins, para-aminobenzoic acid, biotin, amino acids, choline, and copper sulfate. The feed culture medium uses sterile deionized water as a solvent and contains glucose, urea, potassium dihydrogen phosphate, potassium chloride, and calcium chloride. The vitamin is two or more of vitamin B5, vitamin B1, vitamin B6, vitamin B3 or vitamin B2; The amino acid is a mixture of two or more of isoleucine, tyrosine, lysine, or histidine.

[0008] A further improvement to the present invention is as follows: The mass percentages of each component in the substrate culture medium are as follows: 0.09%–0.20% glucose, 0.065%–0.135% magnesium sulfate, 0.014%–0.029% zinc sulfate, 0.011%–0.023% ferrous sulfate, 0.00006%–0.00014% manganese sulfate, 0.000008%–0.000016% potassium iodide, 0.005%–0.013% inositol, 0.0018%–0.0039% vitamin B5, 0.000022%–0.000047% para-aminobenzoic acid, and 0.0000075%–0.00% [unclear - possibly a typo]. 0.0016% biotin, 0.035%–0.075% isoleucine, 0.007%–0.017% lysine, 0.024%–0.048% histidine, 0.00027%–0.00060% vitamin B1, 0.00015%–0.00032% vitamin B6, 0.00019%–0.00038% vitamin B3, 0.000015%–0.000033% vitamin B2, 0.00009%–0.00021% choline, 0.000020%–0.000044% copper sulfate, with the balance being sterile deionized water.

[0009] Preferably, the mass percentage of each component in the substrate culture medium is as follows: 0.14% glucose, 0.1% magnesium sulfate, 0.0206% zinc sulfate, 0.016% ferrous sulfate, 0.000096% manganese sulfate, 0.000012% potassium iodide, 0.00804% inositol, 0.00264% vitamin B5, 0.0000323% para-aminobenzoic acid, 0.0000111% biotin, 0.0504% isoleucine, 0.012% lysine, 0.0336% histidine, 0.000412% vitamin B1, 0.00021% vitamin B6, 0.00027% vitamin B3, 0.0000223% vitamin B2, 0.000147% choline, 0.00003% copper sulfate, with the balance being sterile deionized water.

[0010] Furthermore, the mass percentages of each component in the supplemental culture medium are as follows: 6%–11% glucose, 0.45%–0.85% urea, 0.14%–0.30% potassium dihydrogen phosphate, 0.063%–0.142% potassium chloride, 0.0068%–0.0146% calcium chloride, with the remainder being sterile deionized water.

[0011] Preferably, the mass percentage of each component in the supplemental culture medium is 8% glucose, 0.609% urea, 0.215% potassium dihydrogen phosphate, 0.1% potassium chloride, 0.01% calcium chloride, and the remainder is sterile deionized water.

[0012] A further improvement of the present invention is as follows: The method for fermenting baker's yeast using the above-mentioned culture medium includes the following steps: (1) Treatment of strains: After thawing the frozen yeast strains, inoculate them into YPD liquid medium and shake culture to obtain activated bacterial solution. The activated bacterial solution is then purified by streak plate culture, and plates with good single colony growth are selected as working plates. (2) Preparation of fermenter seed liquid: Select a single colony of yeast on the working plate and inoculate it into the primary seed liquid culture medium and culture it at high speed to obtain the primary seed liquid. Inoculate the primary seed liquid into the secondary seed liquid culture medium and culture it at high speed to obtain the secondary seed liquid. Inoculate the secondary seed liquid into the tertiary seed liquid culture medium and culture it at high speed. After centrifugation, washing and resuspending in sterile water, the fermenter seed liquid is obtained. (3) Fermentation culture: Centrifuge the seed liquid in the fermenter and discard the supernatant. Collect the yeast cells and inoculate them into the fermenter on the bottom culture medium for high-density fermentation. During the fermentation process, stir, control the temperature, adjust the pH value, and add feed medium intermittently. After the nitrogen and phosphorus source ends 1 h, adjust the culture conditions and continue the induction culture. After the culture is completed, centrifuge and collect the yeast cells to obtain low-sugar fresh yeast.

[0013] Furthermore, the plate described in step (1) is placed in an incubator at 30~32℃ and cultured for 36-48 hours.

[0014] Furthermore, the primary seed culture medium, secondary seed culture medium and tertiary seed culture medium mentioned in step (2) are all YPD liquid culture medium.

[0015] Furthermore, in step (2), the temperature for the rotational culture is 30~32℃, the rotation speed is 180~220rpm, and the time is 10~14h.

[0016] Furthermore, in step (3), the high-density fermentation temperature is 30-32℃, the pH is 4.3-4.7, the stirring speed is 700-900 r / min, the ventilation rate is 7-9, the total fermentation time is 18 hours, and feed is added every 1-2 hours during the fermentation process.

[0017] Furthermore, the induction culture conditions described in step (3) are: temperature 35-37 ℃, pH 5.0-5.4, stirring speed 250-350 r / min, ventilation 1-3, and induction time 1-1.5h.

[0018] Compared with the prior art, the present invention has the following beneficial effects: (1) The present invention uses glucose as the main carbon source, which is beneficial to reducing the dependence of low sugar fresh yeast production on molasses source and batch stability; (2) This invention compensates for the lack of auxiliary nutritional factors in the glucose main carbon source system by compounding amino acids, trace elements and vitamins; (3) The present invention combines culture medium compounding with the later induction process, which can improve the fermentation power after storage while maintaining a high initial fermentation power; (4) Under the same fermentation conditions, compared with beet molasses culture medium, the glucose compound culture medium of the present invention can obtain low sugar fresh yeast with fermentation power and storage stability no less than the control system. Attached Figure Description

[0019] Figure 1 This is a schematic diagram comparing the effects of the glucose compound culture medium of the present invention and the beet molasses culture medium used in the prior art in preparing low-sugar fresh yeast. Detailed Implementation

[0020] This invention provides a low-sugar baker's yeast culture medium that can replace molasses, and a method for obtaining fresh yeast for low-sugar dough through high-density fermentation using this culture medium. In specific embodiments, unless otherwise specified, the experimental methods used are conventional methods, and the materials and reagents used are commercially available unless otherwise specified. The invention will be described in detail below with reference to specific embodiments.

[0021] The YPD solid culture medium (mass fraction) used in this invention is: 2% glucose, 1% yeast extract, 2% peptone, and 2% agar, prepared with sterile deionized water and sterilized at 115°C for 20 minutes.

[0022] YPD liquid culture medium (i.e., primary seed culture medium, secondary seed culture medium, and tertiary seed culture medium; mass fraction) is: 2% glucose, 1% yeast extract, and 2% peptone, prepared with sterile deionized water and sterilized at 115℃ for 20 minutes.

[0023] The detection method involved in this invention: (1) Determination of initial fermentation power: After the yeast sample culture is completed, it is placed at 4℃ for 24 h and then the fermentation power is measured. The result is recorded as the initial fermentation power.

[0024] (2) Fermentation power determination after storage: After the fresh yeast cells were cultured, they were stored at 30℃ for 48 h and the fermentation power was determined. The result was recorded as the fermentation power after storage.

[0025] (3) The fermentation power was determined using the standard dough draining method. 16 g of sodium chloride was weighed and dissolved in about 580 mL of distilled water to prepare a salt solution; another 20 g of sodium chloride was weighed, dissolved in water, and then 2 mL of concentrated sulfuric acid was added, followed by dilution to 200 mL with water to prepare the drained liquid. 28 g of wheat flour was weighed, and 0.6 g of fresh yeast sample (calculated based on 30% dry matter content) was added, followed by 14.5 mL of salt solution and rapid mixing. The dough preparation was completed within 5 min, and the final dough temperature was controlled at 30℃±0.2℃. The dough was transferred to a fermentation bottle and placed in a 30℃ constant temperature water bath. Starting from the 8th minute after kneading began, the volume of drained liquid was collected within 1.5 h, and the average value was multiplied by 10 to obtain the fermentation power of the batch of yeast sample, in mL / 1.5 h.

[0026] (4) The intracellular trehalose content was determined by the anthrone sulfuric acid method, and the results were expressed as a percentage of yeast cell dry weight.

[0027] Example 1: Preparation of glucose compound culture medium The substrate culture medium, using sterile deionized water as a solvent, contains the following components by mass fraction: 0.14% glucose, 0.1% magnesium sulfate, 0.0206% zinc sulfate, 0.016% ferrous sulfate, 0.000096% manganese sulfate, 0.000012% potassium iodide, 0.00804% inositol, 0.00264% vitamin B5, 0.0000323% para-aminobenzoic acid, 0.0000111% biotin, 0.0504% leucine, 0.012% lysine, 0.0336% histidine, 0.000412% vitamin B1, 0.00021% vitamin B6, 0.00027% vitamin B3, 0.0000223% vitamin B2, 0.000147% choline, and 0.00003% copper sulfate. In this process, glucose was dissolved in deionized water and sterilized at 115°C for 20 minutes. Other nutrients were prepared into a stock solution using sterile deionized water and then filtered through a 0.22 μm filter membrane for sterilization. The solution was then added to the bottom of the tank under sterile conditions to prepare the culture medium.

[0028] Feeding medium: using sterile deionized water as solvent, containing 8% glucose, 0.609% urea, 0.215% potassium dihydrogen phosphate, 0.1% potassium chloride, and 0.01% calcium chloride by mass fraction. Glucose is dissolved in deionized water and sterilized at 115℃ for 20 min. Other nutrients are prepared with sterile deionized water and then filtered through a 0.22 μm filter membrane for sterilization before use.

[0029] Example 2: Preparation of beet molasses culture medium The substrate culture medium, using sterile deionized water as a solvent, contains 4% glucose, 0.0943% magnesium sulfate, 0.794% sodium chloride, 0.0914% calcium chloride, 0.0000857% nickel chloride, 0.0000286% potassium iodide, 0.00371% zinc sulfate, 0.002% ferrous sulfate, 0.000171% copper sulfate, 0.000114% cobalt chloride, 0.0000123% biotin, 0.000769% vitamin B5, 0.000523% vitamin B1, 0.000246% vitamin B6, 0.0000791% vitamin B2, 0.000432% para-aminobenzoic acid, and 0.0021% choline. Glucose was dissolved in deionized water and sterilized at 115°C for 20 minutes. Other nutrients were prepared using sterile deionized water and then filtered through a 0.22 μm filter membrane for sterilization. The mixture was then added to the bottom of the tank under sterile conditions to prepare the culture medium.

[0030] Feeding medium: using sterile deionized water as solvent, containing 0.14% glucose, 8.51% beet molasses, 0.609% urea, and 0.215% potassium dihydrogen phosphate by mass fraction. Glucose and beet molasses were dissolved in deionized water and sterilized at 115℃ for 20 min. Other nutrients were prepared with sterile deionized water and then filtered through a 0.22 μm filter membrane for sterilization before use.

[0031] Example 3: Preparation of Seed Liquid in Fermenter Yeast strain No. 99, stored at -80 ℃, was taken out and thawed at 4 ℃. 1 mL of bacterial culture was inoculated into 50 / 250 mL of YPD liquid medium and cultured with shaking at 30 ℃ and 200 r / min for 12 h to obtain activated bacterial culture. The activated bacterial culture was purified by streak plating, and single colonies with good growth and normal morphology were picked and inoculated into primary seed culture medium and cultured at 30 ℃ and 200 rpm for 12 h to obtain primary seed culture. 5% (V / V) of primary seed culture was inoculated into secondary seed culture medium and cultured at 30 ℃ and 200 rpm for 12 h to obtain secondary seed culture. 5% (V / V) of secondary seed culture was inoculated into 500 mL of tertiary seed culture medium and cultured at 30 ℃ and 200 rpm for 24 h in a shaker. The cultured seed culture was centrifuged at 8000 rpm for 8 min at 4 ℃, the supernatant was discarded, the bacterial cells were washed 2-3 times with sterile water, and the volume was resuspended in sterile water to 150 mL to obtain seed culture.

[0032] Example 4: Preparation of low-sugar fresh yeast using glucose-based culture medium The glucose-based culture medium described in Example 1 was used for fermentation in a 5 L fermenter, with an actual working volume of 3.5 L. After the bottom culture medium (1.2 L) in the fermenter was sterilized, cooled, and all parameters were adjusted to be stable, 150 mL of seed culture prepared in Example 3 was inoculated under aseptic conditions.

[0033] The initial fermentation conditions were: temperature 30℃, pH 4.5, stirring speed 800 r / min, and aeration rate 8. During fermentation, when the biomass reached 75-90 g / L, fed medium (100 mL) was added intermittently. After the nitrogen and phosphorus sources were depleted for 1 hour, the conditions were adjusted to: temperature 35℃, pH 5.0, stirring speed 300 r / min, and aeration rate 2, and induction culture continued for another 1-1.5 hours. After the culture was completed, the yeast cells were collected by centrifugation to obtain low-sugar fresh yeast.

[0034] Under these conditions, when samples were taken 1 h after nitrogen and phosphorus source depletion, the initial fermentation capacity of the obtained low-sugar fresh yeast reached 1300 mL / 1.5 h, and the fermentation capacity after storage reached 950 mL / 1.5 h. When samples were taken 1.5 h after changing the culture conditions, the initial fermentation capacity still reached 1190 mL / 1.5 h, and the fermentation capacity after storage reached 895 mL / 1.5 h, with an intracellular trehalose content of 22.2%. These results indicate that by using a glucose-based culture medium combined with late-stage induction regulation after nitrogen and phosphorus source depletion, low-sugar fresh yeast with high initial and post-storage fermentation capacities can be obtained.

[0035] Example 5: Preparation of low-sugar fresh yeast from beet molasses culture medium The beet molasses culture medium described in Example 2 was used for fermentation in a 5 L fermenter. Low-sugar fresh yeast was prepared under the same fermentation conditions as in Example 5, and the initial fermentation power, post-storage fermentation power, and intracellular trehalose content were measured.

[0036] The comparison results are shown below. Figure 1 When samples were taken 1 h after the nitrogen and phosphorus source was exhausted, the initial fermentation power of the glucose compound group (1300) and the beet molasses group (1280) was similar, but the fermentation power of the former after storage (950) was significantly higher than that of the latter (880). The trehalose content of the beet molasses group (20.0) was slightly higher than that of the glucose compound group (16.9), proving that the protective substances accumulated faster in the beet molasses system, but this did not translate into an advantage in storage performance. Further comparisons 1.5 h after changing the culture conditions showed that the glucose compound group was superior to the beet molasses group in terms of initial fermentation power (1190), fermentation power after storage (895), and trehalose content (22.2), indicating that after late-stage induction and regulation, the glucose compound system can balance fermentation performance and the accumulation of protective substances.

[0037] The above description of the embodiments is only for illustrating the technical concept and features of the present invention. Its purpose is to enable those skilled in the art to understand the content of the present invention and implement it accordingly. Those skilled in the art can obviously easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative effort. Therefore, the above embodiments should not be used to limit the scope of protection of the present invention. All improvements and modifications made by those skilled in the art based on the disclosure of the present invention without departing from the scope of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A low-sugar baker's yeast culture medium that can replace molasses, characterized in that, The culture medium is a glucose compound culture medium without added molasses, using glucose as the main carbon source. The glucose compound culture medium consists of a base culture medium and a feed culture medium. The base culture medium uses sterile deionized water as a solvent and contains glucose, magnesium sulfate, zinc sulfate, ferrous sulfate, manganese sulfate, potassium iodide, inositol, vitamins, para-aminobenzoic acid, biotin, amino acids, choline, and copper sulfate. The feed culture medium uses sterile deionized water as a solvent and contains glucose, urea, potassium dihydrogen phosphate, potassium chloride, and calcium chloride. The vitamin is two or more of vitamin B5, vitamin B1, choline, vitamin B3 or vitamin B2; The amino acid is a mixture of two or more of isoleucine, tyrosine, lysine, or histidine.

2. The low-sugar baker's yeast culture medium that can replace molasses according to claim 1, characterized in that: The mass percentages of each component in the substrate culture medium are as follows: 0.09%–0.20% glucose, 0.065%–0.135% magnesium sulfate, 0.014%–0.029% zinc sulfate, 0.011%–0.023% ferrous sulfate, 0.00006%–0.00014% manganese sulfate, 0.000008%–0.000016% potassium iodide, 0.005%–0.013% inositol, 0.0018%–0.0039% vitamin B5, 0.000022%–0.000047% para-aminobenzoic acid, and 0.0000075%–0.00% [unclear - possibly a typo]. 0.0016% biotin, 0.035%–0.075% isoleucine, 0.007%–0.017% lysine, 0.024%–0.048% histidine, 0.00027%–0.00060% vitamin B1, 0.00015%–0.00032% vitamin B6, 0.00019%–0.00038% vitamin B3, 0.000015%–0.000033% vitamin B2, 0.00009%–0.00021% choline, 0.000020%–0.000044% copper sulfate, with the balance being sterile deionized water.

3. The low-sugar baker's yeast culture medium that can replace molasses according to claim 2, characterized in that: The mass percentages of each component in the substrate culture medium are as follows: 0.14% glucose, 0.1% magnesium sulfate, 0.0206% zinc sulfate, 0.016% ferrous sulfate, 0.000096% manganese sulfate, 0.000012% potassium iodide, 0.00804% inositol, 0.00264% vitamin B5, 0.0000323% para-aminobenzoic acid, 0.0000111% biotin, 0.0504% isoleucine, 0.012% lysine, 0.0336% histidine, 0.000412% vitamin B1, 0.00021% vitamin B6, 0.00027% vitamin B3, 0.0000223% vitamin B2, 0.000147% choline, 0.00003% copper sulfate, with the remainder being sterile deionized water.

4. The low-sugar baker's yeast culture medium that can replace molasses according to claim 1, characterized in that: The mass percentages of each component in the feed culture medium are as follows: 6%–11% glucose, 0.45%–0.85% urea, 0.14%–0.30% potassium dihydrogen phosphate, 0.063%–0.142% potassium chloride, 0.0068%–0.0146% calcium chloride, with the remainder being sterile deionized water.

5. A low-sugar baker's yeast culture medium that can replace molasses according to claim 4, characterized in that: The feed culture medium contains the following components by mass percentage: 8% glucose, 0.609% urea, 0.215% potassium dihydrogen phosphate, 0.1% potassium chloride, 0.01% calcium chloride, and the remainder is sterile deionized water.

6. A method for fermenting baker's yeast using the culture medium according to any one of claims 1 to 5, characterized in that, Includes the following steps: (1) Treatment of strains: After thawing the frozen yeast strains, inoculate them into YPD liquid medium and shake culture to obtain activated bacterial solution. The activated bacterial solution is then purified by streak plate culture, and plates with good single colony growth are selected as working plates. (2) Preparation of fermenter seed liquid: Select a single colony of yeast on the working plate and inoculate it into the primary seed liquid culture medium and culture it at high speed to obtain the primary seed liquid. Inoculate the primary seed liquid into the secondary seed liquid culture medium and culture it at high speed to obtain the secondary seed liquid. Inoculate the secondary seed liquid into the tertiary seed liquid culture medium and culture it at high speed. After centrifugation, washing and resuspending in sterile water, the fermenter seed liquid is obtained. (3) Fermentation culture: Centrifuge the seed liquid in the fermenter and discard the supernatant. Collect the yeast cells and inoculate them into the fermenter on the bottom culture medium for high-density fermentation. During the fermentation process, stir, control the temperature, adjust the pH value, and add supplementary culture medium intermittently. After the nitrogen and phosphorus source ends 1 hour, adjust the culture conditions and continue the induction culture. After the culture is completed, centrifuge and collect the yeast cells to obtain low-sugar fresh yeast.

7. The fermentation method according to claim 6, characterized in that: The plate described in step (1) is placed in an incubator at 30~32℃ and cultured for 36-48 hours.

8. The fermentation method according to claim 6, characterized in that: The primary seed culture medium, secondary seed culture medium, and tertiary seed culture medium mentioned in step (2) are all YPD liquid culture medium; And / or, the temperature for the rotational culture is 30~32℃, the rotation speed is 180~220rpm, and the time is 10~14h.

9. The fermentation method according to claim 6, characterized in that: The high-density fermentation in step (3) is carried out at a temperature of 30-32℃, a pH of 4.3-4.7, a stirring speed of 700-900 r / min, a ventilation rate of 7-9, and a total fermentation time of 18 hours. During the fermentation process, feed is added every 1-2 hours.

10. The fermentation method according to claim 6, characterized in that: The conditions for induction culture described in step (3) are: temperature 35-37 ℃, pH 5.0-5.4, stirring speed 250-350 r / min, ventilation 1-3, and induction time 1-1.5h.

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