Yeast culture medium as well as fermentation culture method and application method thereof

Abstract

The invention discloses a yeast culture medium as well as a fermentation culture method and application thereof, and belongs to the field of microbial fermentation. The saccharomycetes culture medium is prepared from the following raw materials in percentage by mass: 0.2 to 1 percent of wheat dry alcohol lees, 0.1 to 1 percent of yeast extract powder, 1 to 8 percent of molasses, 1 to 3 percent of methanol, 0.1 to 0.9 percent of disodium hydrogen phosphate, 0.1 to 1 percent of ammonium citrate, 0.1 to 1 percent of dipotassium phosphate, 0.005 to 0.05 percent of magnesium sulfate, 0.002 to 0.015 percent of manganese chloride and the balance of water. The culture medium is reasonable in formula, under the condition that glucose and peptone are not added and only a small amount of yeast extract powder is added, thallus growth can still be effectively promoted in the same fermentation period, and compared with a traditional saccharomycetes culture medium, the thallus density and the acid yield in a bacterial solution are remarkably improved, and the industrial production cost is remarkably reduced.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation, and specifically relates to a yeast culture medium and a fermentation culture method and application thereof. Background Art

[0002] In the production process of organic acids by microbial fermentation, as its own acidic products accumulate, the pH of the fermentation liquid continues to decrease, seriously affecting the metabolic activity and production efficiency of the cells. Therefore, lactic acid fermentation is generally produced by the calcium salt method, but it has problems such as high production cost, many process links, large amount of calcium sulfate solid waste, and serious environmental pollution. It is reported that many yeasts can grow in harsh environments with a pH below 2.0. There is no need to add alkaline neutralizers to maintain the fermentation pH during the fermentation process, and no calcium sulfate solid waste will be generated. This is in line with the national sustainable development concept and has certain social and economic benefits.

[0003] The formula of fermentation medium is one of the core factors that determine the quality of microbial fermentation. When the medium formula ratio is not suitable or the elements are not selected well, it will have a negative impact on the growth and metabolism of the bacteria. The low growth density of the bacteria will lead to metabolic imbalance, which is not conducive to product accumulation. On the contrary, if the medium formula composition is appropriate, it will also promote the growth of the bacteria and improve the activity and vitality of the bacteria. The traditional yeast culture medium YPD (yeast extract powder peptone glucose medium) contains a large amount of yeast extract powder and peptone, and the production cost is relatively high. Summary of the invention

[0004] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a yeast culture medium and a fermentation culture method and application thereof, which solve the problems in the prior art.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A yeast culture medium comprises the following raw materials in percentage by mass: 0.2-1% of wheat dry alcohol grains, 0.1-1% of yeast extract powder, 1-8% of molasses, 1-3% of methanol, 0.1-0.9% of disodium hydrogen phosphate, 0.1-1% of ammonium citrate, 0.1-1% of dipotassium hydrogen phosphate, 0.005-0.05% of magnesium sulfate, 0.002-0.015% of manganese chloride, and the balance is water.

[0007] The use of the above-mentioned yeast culture medium in the preparation of lactic acid.

[0008] A yeast fermentation culture method, using the above yeast culture medium, comprises the following steps:

[0009] S1, inoculate the yeast into a shake flask filled with YPD medium for activation culture to obtain a first-level seed solution;

[0010] S2, inoculating the primary seed solution into a shake flask filled with YPD medium for expansion to obtain a secondary seed solution;

[0011] S3, inoculating the secondary seed liquid into the yeast culture medium for fermentation culture.

[0012] Furthermore, in S1, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, and the culture time is 20h.

[0013] Furthermore, in S1, the inoculation amount of yeast is 2%.

[0014] Further, in S2, the culture temperature is 30-35°C, the rotation speed is 200-220 rpm, and the culture time is 20 h;

[0015] Furthermore, in S2, the inoculation amount of the primary seed solution is 2%.

[0016] Furthermore, in S3, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, and the culture time is 48h.

[0017] Furthermore, in S3, the inoculation amount of the secondary seed solution is 2%.

[0018] Beneficial effects of the present invention:

[0019] 1. The raw materials used in the present invention are low-priced, and expensive raw materials such as peptone, glucose and a large amount of yeast extract powder in traditional yeast culture medium are not required. The cost of the culture medium of the present invention is greatly reduced compared with the traditional culture medium.

[0020] 2. The culture medium provided by the present invention has excellent practicality. Compared with the traditional yeast culture medium, the cell density and acid production are improved to a certain extent, and the industrial production cost is significantly reduced;

[0021] 3. The culture medium provided by the present invention is close to, reaches or even exceeds the level of traditional culture medium, and can well replace the more expensive traditional culture medium. DETAILED DESCRIPTION

[0022] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solution of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0023] A yeast culture medium comprises the following raw materials in percentage by mass: 0.2-1% of wheat dry alcohol grains, 0.1-1% of yeast extract powder, 1-8% of molasses, 1-3% of methanol, 0.1-0.9% of disodium hydrogen phosphate, 0.1-1% of ammonium citrate, 0.1-1% of dipotassium hydrogen phosphate, 0.005-0.05% of magnesium sulfate, 0.002-0.015% of manganese chloride, and the balance is water.

[0024] The yeast fermentation culture method comprises the following steps:

[0025] S1, inoculating yeast into a shake flask filled with YPD medium for activation culture to obtain a first-level seed solution; wherein, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, the culture time is 20h, and the inoculation amount of the yeast is 2%.

[0026] S2, inoculating the primary seed solution into a shaking flask filled with YPD culture medium to obtain a secondary seed solution; wherein the culture temperature is 30-35°C, the rotation speed is 200-220rpm, the culture time is 20h, and the inoculation amount of the primary seed solution is 2%.

[0027] S3, inoculating the secondary seed liquid into the yeast culture medium for fermentation culture; wherein the culture temperature is 30-35° C., the rotation speed is 200-220 rpm, and the culture time is 48 hours; and the inoculation amount of the secondary seed liquid is 2%.

[0028] The scheme of the present invention is specifically described below by the following examples and comparative examples, wherein all raw material information (manufacturer information) in the examples and comparative examples is as follows:

[0029] Wheat distillers grains, Huamao (Shandong) Biotechnology Co., Ltd.

[0030] Yeast extract powder, Angel Yeast Co., Ltd.;

[0031] Molasses, Jiangsu Caiwei Biotechnology Co., Ltd.;

[0032] Methanol, Sinopharm Group Co., Ltd.;

[0033] Disodium hydrogen phosphate, Sinopharm Group Pharmaceutical Co., Ltd.;

[0034] Ammonium citrate, Sinopharm Group Pharmaceutical Co., Ltd.;

[0035] Potassium hydrogen phosphate, Sinopharm Group Co., Ltd.;

[0036] Magnesium sulfate, Sinopharm Group Pharmaceutical Co., Ltd.;

[0037] Manganese chloride, Sinopharm Group Pharmaceutical Co., Ltd.;

[0038] Peptone: OXOID;

[0039] Glucose: Xiwang Sugar Co., Ltd.;

[0040] All reagents and raw materials are commercially available.

[0041] Example 1

[0042] In this embodiment, the yeast culture medium includes the following raw materials in weight percentage:

[0043] 0.6% wheat dry alcohol grains, 0.1% yeast extract powder, 4% molasses, 1% methanol, 0.5% disodium hydrogen phosphate, 0.4% ammonium citrate, 0.5% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.015% manganese chloride, and the balance is water.

[0044] The yeast fermentation culture method comprises the following steps:

[0045] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0046] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0047] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0048] Example 2

[0049] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0050] 0.4% wheat dry grains, 0.5% yeast extract powder, 6% molasses, 1.5% methanol, 0.7% disodium hydrogen phosphate, 0.2% ammonium citrate, 0.8% dipotassium hydrogen phosphate, 0.01% magnesium sulfate, 0.012% manganese chloride, and the balance is water.

[0051] The yeast fermentation culture method comprises the following steps:

[0052] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0053] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0054] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0055] Example 3

[0056] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0057] 0.8% wheat dry grains, 0.3% yeast extract powder, 2% molasses, 2% methanol, 0.35% disodium hydrogen phosphate, 0.6% ammonium citrate, 0.3% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.008% manganese chloride, and the balance is water.

[0058] The yeast fermentation culture method comprises the following steps:

[0059] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0060] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0061] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0062] Example 4

[0063] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0064] 0.2% wheat dry grains, 1% yeast extract powder, 8% molasses, 2.5% methanol, 0.9% disodium hydrogen phosphate, 0.1% ammonium citrate, 1% dipotassium hydrogen phosphate, 0.005% magnesium sulfate, 0.005% manganese chloride, and the balance is water.

[0065] The yeast fermentation culture method comprises the following steps:

[0066] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0067] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0068] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0069] Example 5

[0070] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0071] 1% wheat dry grains, 0.1% yeast extract powder, 1% molasses, 3% methanol, 0.1% disodium hydrogen phosphate, 1% ammonium citrate, 0.1% dipotassium hydrogen phosphate, 0.05% magnesium sulfate, 0.002% manganese chloride, and the balance is water.

[0072] The yeast fermentation culture method comprises the following steps:

[0073] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0074] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0075] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0076] Example 6

[0077] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0078] 0.2% wheat dry alcohol grains, 0.1% yeast extract powder, 4% molasses, 1% methanol, 0.5% disodium hydrogen phosphate, 0.4% ammonium citrate, 0.5% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.015% manganese chloride, and the balance is water.

[0079] The yeast fermentation culture method comprises the following steps:

[0080] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0081] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0082] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0083] Example 7

[0084] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0085] 0.4% wheat dry grains, 0.1% yeast extract powder, 4% molasses, 1% methanol, 0.5% disodium hydrogen phosphate, 0.4% ammonium citrate, 0.5% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.015% manganese chloride, and the balance is water.

[0086] The yeast fermentation culture method comprises the following steps:

[0087] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0088] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0089] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0090] Example 8

[0091] In this embodiment, the yeast culture medium includes the following raw materials in percentage by weight:

[0092] 0.8% wheat dry grains, 0.1% yeast extract powder, 4% molasses, 1% methanol, 0.5% disodium hydrogen phosphate, 0.4% ammonium citrate, 0.5% dipotassium hydrogen phosphate, 0.03% magnesium sulfate, 0.015% manganese chloride, and the balance is water.

[0093] The yeast fermentation culture method comprises the following steps:

[0094] (1) The yeast stored in a -80°C refrigerator was naturally thawed in advance, and transferred to a shake flask filled with YPD medium at a 2% inoculation rate for activation culture at a temperature of 30°C and a rotation speed of 200 rpm for 20 h to obtain a primary seed solution.

[0095] (2) The first-level seed solution obtained in step (1) was transferred to a shake flask filled with YPD medium at an inoculum size of 2% for expansion culture at a temperature of 30° C. and a rotation speed of 200 rpm for 20 h to obtain a second-level seed solution.

[0096] (3) The secondary seed liquid was transferred to the yeast culture medium at a 2% inoculation rate, and the fermentation was terminated after culturing for 48 hours.

[0097] Comparative Example 1

[0098] The only difference from Example 1 is that the yeast culture medium in step (3) is replaced by YPD culture medium.

[0099] Comparative Example 2

[0100] The only difference from Example 1 is that no wheat distillers grains are added to the yeast culture medium.

[0101] Comparative Example 3

[0102] The only difference from Example 1 is that no molasses is added to the yeast culture medium.

[0103] Experimental testing

[0104] The products after fermentation in Examples 1-8 and Comparative Examples 1-3 were measured, and the cell density and lactic acid concentration at the end of fermentation were measured. The specific results are shown in Table 1.

[0105] Table 1 Detection results of fermentation products of Examples and Comparative Examples

[0106] Colony count (cfu / mL) Lactic acid concentration (g / L) Example 1 <![CDATA[9.32×10 7 ]]> 25.2 Example 2 <![CDATA[6.34×10 7 ]]> 12.4 Example 3 <![CDATA[5.66×10 7 ]]> 11.2 Example 4 <![CDATA[4.38×10 7 ]]> 9.6 Example 5 <![CDATA[2.74×10 7 ]]> 2.2 Example 6 <![CDATA[7.72×10 7 ]]> 21.4 Example 7 <![CDATA[8.34×10 7 ]]> 22.8 Example 8 <![CDATA[9.64×10 7 ]]> 25.6 Comparative Example 1 <![CDATA[7.50×10 7 ]]> 20.6 Comparative Example 2 <![CDATA[4.06×10 7 ]]> 9.8 Comparative Example 3 <![CDATA[1.60×10 7 ]]> 1.8

[0107] According to the data of the examples and comparative examples in Table 1, it can be seen that the yeast culture medium described in the present application can increase the cell density and effectively improve the cell growth rate and cell activity within the same fermentation cycle;

[0108] From the data of Examples 6-8 and Example 1, it can be seen that as the amount of wheat dry alcohol grains added increases, the cell density and the content of metabolite lactic acid also increase, indicating that wheat dry alcohol grains play a vital role in yeast fermentation; in addition, through the detection of the concentration of metabolite lactic acid, it was found that the lactic acid concentration in the bacterial liquid using the yeast culture medium of the example of the present application was higher;

[0109] In addition, the culture medium data of the embodiment and comparative example 1 show that, when glucose and peptone are not used and only a very small amount of yeast extract powder is added, the cell density can be effectively increased and the industrial production cost can be greatly reduced by adjusting the composition ratio of the culture medium.

[0110] In the description of this specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0111] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A yeast culture medium, characterized in that The invention comprises the following raw materials in percentage by mass: 0.2-1% of wheat dry alcohol grains, 0.1-1% of yeast extract powder, 1-8% of molasses, 1-3% of methanol, 0.1-0.9% of disodium hydrogen phosphate, 0.1-1% of ammonium citrate, 0.1-1% of dipotassium hydrogen phosphate, 0.005-0.05% of magnesium sulfate, 0.002-0.015% of manganese chloride, and the balance is water.

2. Use of the yeast culture medium according to claim 1 in the preparation of lactic acid.

3. A yeast fermentation culture method, using the yeast culture medium according to claim 1, characterized in that: The following steps are involved: S1, inoculating yeast into a shake flask filled with YPD medium for activation culture to obtain a first-level seed solution; S2, inoculating the primary seed solution into a shake flask filled with YPD medium for expansion to obtain a secondary seed solution; S3, inoculating the secondary seed liquid into the yeast culture medium for fermentation culture.

4. A yeast fermentation and culturing method according to claim 3, characterized in that: In S1, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, and the culture time is 20h.

5. A yeast fermentation and culturing method according to claim 3, characterized in that: In S1, the yeast inoculation amount was 2%.

6. A yeast fermentation and culturing method according to claim 3, characterized in that: In S2, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, and the culture time is 20h.

7. The yeast fermentation and culturing method according to claim 3, characterized in that: In S2, the inoculation amount of the primary seed solution was 2%.

8. The yeast fermentation and culturing method according to claim 3, characterized in that: In S3, the culture temperature is 30-35°C, the rotation speed is 200-220rpm, and the culture time is 48h.

9. A yeast fermentation and culturing method according to claim 3, characterized in that: In S3, the inoculation amount of the secondary seed solution was 2%.