Cinnamon essential oil tolerant brewer's yeast and its domestication method and application

Abstract

The present invention discloses a cinnamon essential oil-tolerant brewer's yeast and a domestication method and application thereof, and belongs to the field of microbial engineering technology. The cinnamon essential oil-tolerant brewer's yeast of the present invention is classified and named Saccharomyces cerevisiae BM6313, which has been deposited in the China Center for Type Culture Collection, with a deposit number of: CCTCC NO: M20221782, a deposit date of: November 14, 2022, and a deposit address of: Wuhan University, China. The results of fermenting cinnamon distillation residues using brewer's yeast BM6313 showed that the crude protein content in the cinnamon distillation residues before fermentation was 3.76%; after adding brewer's yeast BM6313, the crude protein content in the fermented feed of the cinnamon distillation residues was 14.46%.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial engineering, and more specifically relates to a cinnamon essential oil-tolerant brewer's yeast and a domestication method and application thereof. Background Art

[0002] The main components of cinnamon essential oil are aromatic bioactive substances such as cinnamaldehyde, linalool and eugenol, which have inhibitory effects on a variety of microorganisms, especially some pathogenic bacteria, such as Salmonella typhi, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, Listeria, Campylobacter jejuni, Klebsiella pneumoniae and Mycobacterium tuberculosis. It is currently widely used in the field of feed additives, which has provided support for reducing or replacing the use of antibiotics, improving the nutritional value of animal feed, strengthening feed utilization, enhancing animal production performance, improving the quality of animal products, and promoting food safety in my country.

[0003] After cinnamon is extracted with steam, some biologically active substances still remain in the distillation residue obtained, which has the potential to prepare "antibiotic replacement" and "antibiotic reduction" feed additives. However, the branches and leaves are mainly lignocellulose, which is difficult to digest and utilize, and its protein content is low and its nutritional value is low. Adding a large amount of cinnamon distillation residue is not conducive to the healthy growth of animals. Fermentation by adding microorganisms with high protein content such as brewer's yeast is an effective means to increase the protein content of fiber feed raw materials and improve the amino acid ratio. However, experiments have shown that cinnamon essential oil has different degrees of damage to the fluidity of most microbial cell membranes, and its antibacterial effect is broad-spectrum. Therefore, it is difficult to obtain ideal results by adding ordinary brewer's yeast to ferment cinnamon distillation residues, and adding mixed microbial agents or enzyme preparations not only increases the complexity of the fermentation process, but also inevitably increases the cost of feed additive preparation. Summary of the invention

[0004] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a cinnamon essential oil tolerant brewer's yeast. Another technical problem to be solved by the present invention is to provide a method for taming cinnamon essential oil tolerant brewer's yeast, which is used to screen brewer's yeast with good tolerance to cinnamon essential oil. Another technical problem to be solved by the present invention is to provide an application of cinnamon essential oil tolerant brewer's yeast.

[0005] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0006] A cinnamon essential oil-tolerant brewer's yeast, classified and named Saccharomyces cerevisiaeBM6313, has been deposited in the China Center for Type Culture Collection, with a deposit number of CCTCC NO: M20221782, a deposit date of November 14, 2022, and a deposit address of Wuhan University, China.

[0007] The domestication method of cinnamon essential oil-tolerant Saccharomyces cerevisiae is as follows: the original starting strain Saccharomyces cerevisiae DSM1334 is placed in a YPD medium containing Triton X-100 and cinnamon essential oil, and the culture is repeated for 5 rounds, and the cinnamon essential oil-tolerant Saccharomyces cerevisiae BM6313 is obtained.

[0008] The formula of the YPD medium is 20 g / L peptone, 10 g / L yeast extract, and 10 g / L glucose.

[0009] The mass volume concentration of the Triton X-100 is 8 g / L.

[0010] The mass volume concentration of the cinnamon essential oil is 40-90 mg / L.

[0011] The mass volume concentration of the cinnamon essential oil is 90 mg / L.

[0012] The domestication method of cinnamon essential oil tolerant Saccharomyces cerevisiae comprises the following specific steps: taking 0.05 mL of the glycerol frozen solution of the original starting strain Saccharomyces cerevisiae DSM1334, inoculating it into a 250 mL conical flask containing 50 mL of freshly prepared YPD medium; placing the conical flask in a shaker at 28°C and culturing it at OD 600 =2.0 to obtain an activated original Saccharomyces cerevisiae DSM1334 bacterial solution; 150 μL of freshly prepared YPD medium was added to 0.02 mL of the activated original Saccharomyces cerevisiae DSM1334 bacterial solution and mixed to prepare a diluted original Saccharomyces cerevisiae DSM1334 bacterial solution; 0.05 mL of the diluted original Saccharomyces cerevisiae DSM1334 bacterial solution and 0.05 mL of YPD medium containing 8 g / L Triton X-100 and 90 mg / L cinnamon essential oil were added to a 96-well plate, and the plate was placed in a 28°C incubator for 48 h, and the acclimation and screening were repeated for 5 rounds to finally obtain the cinnamon essential oil-tolerant Saccharomyces cerevisiae BM6313.

[0013] The preservation method of cinnamon essential oil tolerant brewer's yeast is to inoculate a single colony of brewer's yeast BM6313 into YPD medium, place it in a shaker at a constant temperature of 28°C and a speed of 180 r / min to culture the cells until the optical density D 600 value = 2.0, to prepare the activated bacterial solution of Saccharomyces cerevisiae BM6313; 0.8 mL of the activated bacterial solution of Saccharomyces cerevisiae BM6313 and 0.8 mL of a 40% sterile glycerol aqueous solution were added to a 2 mL cryovial, mixed evenly, and stored in a -80°C environment.

[0014] Application of cinnamon essential oil-tolerant Saccharomyces cerevisiae in improving the protein content of fiber raw materials.

[0015] Application of cinnamon essential oil-tolerant Saccharomyces cerevisiae in increasing the protein content of fermentation products.

[0016] Compared with the prior art, the present invention has the following beneficial effects:

[0017] 1) The cinnamon essential oil-tolerant brewer's yeast of the present invention is classified and named Saccharomyces cerevisiaeBM6313, which has been deposited in the China Center for Type Culture Collection with a deposit number of CCTCC NO: M20221782, a deposit date of November 14, 2022, and a deposit address of Wuhan University, China.

[0018] 2) The cinnamon essential oil tolerant Saccharomyces cerevisiae BM6313 of the present invention is obtained by placing the original starting strain Saccharomyces cerevisiae DSM1334 in a YPD medium containing Triton X-100 and cinnamon essential oil and repeatedly acclimating and screening for 5 rounds. After 20 passages, the Saccharomyces cerevisiae BM6313 has good genetic stability in tolerance to cinnamon essential oil added in an amount of 20 mg / L by mass volume concentration. Compared with the original starting strain Saccharomyces cerevisiae DSM1334, the OD of the strain BM6313 after 20 passages is 2.34~2.73 %. 600 The value increased by 357%-374%; the strain after 20 passages had good genetic stability in tolerance to cinnamon essential oil added at a mass volume concentration of 45 mg / L. Compared with the original starting strain Saccharomyces cerevisiae DSM1334, the OD of the strain BM6313 after 20 passages was 600 The values ​​increased by 1251%-1396%.

[0019] 3) The results of fermenting cinnamon distillation residues with brewer's yeast BM6313 showed that the crude protein content of the cinnamon distillation residues before fermentation was 3.76%; after adding brewer's yeast BM6313, the crude protein content of the fermented feed of cinnamon distillation residues was 14.46%. DETAILED DESCRIPTION

[0020] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0021] The original starting strain Saccharomyces cerevisiae DSM1334 used in the present invention was purchased from the DSMZ strain bank in Germany.

[0022] The formula of YPD medium is: peptone 20g / L, yeast extract 10g / L, glucose 10g / L.

[0023] Example 1

[0024] 1. Take 0.05 mL of the glycerol frozen stock of the original starting strain Saccharomyces cerevisiae DSM1334 and inoculate it into a 250 mL conical flask containing 50 mL of freshly prepared YPD medium; place the conical flask in a shaker at 28°C and culture it until the OD 600 =2.0, and the activated original Saccharomyces cerevisiae DSM1334 bacterial liquid was obtained.

[0025] 2. Add 150 μL of freshly prepared YPD medium to 0.02 mL of activated original Saccharomyces cerevisiae DSM1334 bacterial solution and mix well to prepare a diluted original Saccharomyces cerevisiae DSM1334 bacterial solution.

[0026] 3. Add 0.05 mL of diluted original Saccharomyces cerevisiae DSM1334 bacterial solution and 0.05 mL of YPD medium containing Triton X-100 (mass volume concentration of 8 g / L) and cinnamon essential oil (mass volume concentration of 40 mg / L, 50 mg / L, 60 mg / L, 70 mg / L, 80 mg / L, 90 mg / L) to the 96-well plate. At this time, the mass volume concentration of Triton X-100 in each well of the 96-well plate is 4 g / L, and the mass volume concentrations of cinnamon essential oil are 20 mg / L, 25 mg / L, 30 mg / L, 35 mg / L, 40 mg / L and 45 mg / L, respectively. The 96-well plate is placed in a 28°C incubator for static culture for 48 hours. Repeat acclimatization and screening for 5 rounds under each cinnamon essential oil mass fraction gradient, and detect the growth of the bacteria. After detecting the growth of the bacteria, select OD 600 For the well with the highest value, take 0.06 mL of bacterial solution and dilute it 100-fold to 6 mL using newly prepared YPD medium.

[0027] The results of domestication showed that by adding Triton X-100 to the YPD medium and cinnamon essential oil to work synergistically, the quality gradient of cinnamon essential oil was gradually increased, forming a stable evolutionary pressure on yeast cells. After multiple rounds of domestication, the best growth state and the highest tolerance to cinnamon essential oil were obtained under the conditions of adding cinnamon essential oil at a mass volume concentration of 45 mg / L and Triton X-100 at a mass volume concentration of 4 g / L. The cinnamon essential oil-tolerant brewer's yeast was classified and named Saccharomyces cerevisiae BM6313, which has been deposited in the China Center for Type Culture Collection with a deposit number of: CCTCC NO: M20221782, deposit date: November 14, 2022, and deposit address: Wuhan University, China.

[0028] 4. Low temperature storage

[0029] A single colony of Saccharomyces cerevisiae BM6313 was inoculated into YPD medium and cultured in a shaker at 28°C and 180 r / min until the optical density of the bacteria reached D 600 value = 2.0, to prepare the activated bacterial solution of Saccharomyces cerevisiae BM6313; 0.8 mL of the activated bacterial solution of Saccharomyces cerevisiae BM6313 and 0.8 mL of a 40% sterile glycerol aqueous solution were added to a 2 mL cryovial, mixed evenly, and stored in a -80°C environment.

[0030] Example 2

[0031] 1. Stable propagation of Saccharomyces cerevisiae BM6313

[0032] Use a sterile inoculating loop to dip into the bacterial liquid of cinnamon essential oil-tolerant brewer's yeast BM6313, streak it on a plate containing YPD solid culture, and then invert the plate in a constant temperature incubator at 28°C for 48 hours; randomly select 10 single colonies from the plate of brewer's yeast BM6313, inoculate them into YPD liquid culture medium respectively, and after 20 passages under conditions without evolutionary pressure, use a sterile inoculating loop to dip into the bacterial liquid of brewer's yeast BM6313-201, BM6313-202, BM6313-203, BM6313-204 and BM6313-205 respectively, streak the above bacterial liquids onto plates containing YPD solid culture, invert the plates in a constant temperature incubator at 28°C, culture for 48 hours, and randomly select 1 single colony.

[0033] 2. Test on tolerance of cinnamon essential oil to brewer's yeast

[0034] Use a sterile inoculation loop to dip the bacterial liquid of the original starting strain yeast DSM1334, streak on a plate containing YPD solid culture, and then invert the plate in a 28°C constant temperature incubator for 48 hours; randomly select a single colony from the plate of the original starting strain yeast DSM1334, inoculate the single colony of the starting strain yeast DSM1334 and the single colonies of brewer's yeast BM6313-201, BM6313-202, BM6313-203, BM6313-204 and BM6313-205 into YPD liquid culture medium, place it in a constant temperature shaker at 28°C for shaking at 180 rpm, and wait until the optical density of the bacteria D 600 The culture was stopped after the values ​​increased to 2.0; 0.06 mL of bacterial solution was taken and diluted 100 times to 6 mL using the newly prepared YPD medium; 0.05 mL of the diluted bacterial solution and 0.05 mL of YPD medium containing cinnamon essential oil (mass volume concentration of 20 mg / L and 45 mg / L) were added to a 96-well plate, and 3 groups of parallel treatments were set for each treatment. After 48 hours of culture, the optical density OD of the bacteria was measured 600 value.

[0035] The results are shown in Table 1. The original starting strains of Saccharomyces cerevisiae DSM1334, Saccharomyces cerevisiae BM6313-201, BM6313-202, BM6313-203, BM6313-204 and BM6313-205 were cultured under the same conditions. The strains after 20 passages had good genetic stability to the tolerance of cinnamon essential oil added at a mass volume concentration of 20 mg / L. Compared with the original starting strain of Saccharomyces cerevisiae DSM1334, the OD of the strain BM6313 after 20 passages was 600 The value increased by 357%-374%.

[0036] The original starting strains of Saccharomyces cerevisiae DSM1334, Saccharomyces cerevisiae BM6313-201, BM6313-202, BM6313-203, BM6313-204 and BM6313-205 were cultured under the same conditions. The strains after 20 passages had good genetic stability to the tolerance of cinnamon essential oil added at a mass volume concentration of 45 mg / L. Compared with the original starting strain of Saccharomyces cerevisiae DSM1334, the OD of the strain BM6313 after 20 passages was 600 The values ​​increased by 1251%-1396%.

[0037] In summary, the brewer's yeast BM6313 obtained through multiple rounds of gradient domestication is not only tolerant to high concentrations of cinnamon essential oil, but also has good genetic stability for this target trait.

[0038] Table 1 Stability test of cinnamon essential oil tolerant Saccharomyces cerevisiae

[0039]

[0040] Example 3

[0041] The cinnamon distillation residue is crushed to a particle size of 0.85mm-1.4mm; molasses with a pH of 6.5-7.5 (the amount added is 10% of the weight of the cinnamon distillation residue powder) and brewer's yeast BM6313 (the water content is about 80% based on the weight of the wet bacteria, and the amount added is 5% of the weight of the cinnamon distillation residue powder) are added to the crushed cinnamon distillation residue and mixed, and water is added to the moisture content of 50%; the mixed material is placed at 28°C for aerobic fermentation for 96 hours to obtain cinnamon distillation residue fermented feed. The pre-fermentation material and fermentation product are tested by the Kjeldahl nitrogen method (crude protein content) according to the national standard GB T6432-2018.

[0042] The test results show that the crude protein content of cinnamon distillation residue before fermentation is 3.76%; after adding brewer's yeast BM6313, the crude protein content of cinnamon distillation residue fermented feed is 14.46%. It can be seen that cinnamon distillation residue fermented feed is suitable for use as an additive in the feed industry. It shows application potential in "antibiotic reduction" and "antibiotic replacement" and can also improve the problem of low protein content in fiber raw materials.

[0043] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A cinnamon essential oil tolerant brewer's yeast, which is classified and named as Saccharomyces cerevisiae ( Saccharomyces cerevisiae )BM6313, has been deposited in the China Center for Type Culture Collection, with the deposit number: CCTCC NO: M20221782, the deposit date: November 14, 2022, and the deposit address: Wuhan University, China.

2. The method for preserving cinnamon essential oil tolerant brewer's yeast according to claim 1, characterized in that: A single colony of Saccharomyces cerevisiae BM6313 was inoculated into YPD medium and cultured in a shaker at 28°C and 180 r / min until the optical density of the bacteria was D 600 value = 2.0, to prepare the activated bacterial solution of Saccharomyces cerevisiae BM6313; 0.8 mL of the activated bacterial solution of Saccharomyces cerevisiae BM6313 and 0.8 mL of a 40% sterile glycerol aqueous solution were added to a 2 mL cryovial, mixed evenly, and stored in a -80°C environment.

3. Use of the cinnamon essential oil-tolerant Saccharomyces cerevisiae according to claim 1 in increasing the protein content in cinnamon distillation residues.

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