A Saccharomyces cerevisiae that can ferment soybean molasses and its application

Abstract

The present invention relates to a Saccharomyces cerevisiae strain that can utilize soybean molasses for fermentation and its application. This Saccharomyces cerevisiae strain SC-HKB100 was deposited at the China Center for Type Culture Collection on September 30, 2019, with the deposit number CCTCC NO: M 2019769. Under specific conditions, Saccharomyces cerevisiae strain SC-HKB100 can fully ferment using soybean molasses as a sugar source, effectively utilize sucrose, raffinose, stachyose, etc. in soybean molasses, and reduce antinutritional factors such as saponins in soybean molasses. Compared with traditional fermentation sugar sources such as beet molasses and sucrose molasses, it can significantly reduce the production cost of the yeast fermentation industry, and the fermented product has a high protein content and rich amino acid nutrition, and can be used as an ideal high-quality protein raw material.

Description

Technical Field

[0001] The present invention relates to the technical field of fungi, and particularly relates to a Saccharomyces cerevisiae that can utilize soybean molasses for fermentation and its application. Background Art

[0002] Soybean molasses is a by-product of the production of soy protein concentrate, which is brownish-red and has a strong sweet taste. Its main components are sugars (soybean oligosaccharides), proteins, phospholipids, ash, and phytochemical components such as saponins, isoflavones, and organic acids. Soybean molasses is widely sourced and inexpensive. It is reported that more than 0.34 tons of soybean molasses will be obtained for every 1 ton of alcohol-processed soy protein concentrate produced. However, due to the presence of components such as oligosaccharides (such as raffinose, stachyose, etc.) and saponins in soybean molasses, conventional industrial yeasts and other microorganisms cannot fully utilize its sugars; if directly added to livestock and poultry feed, a large amount of oligosaccharides cannot be digested in the animal digestive tract and will be decomposed by microbial fermentation to produce CH 4 、H 2 、CO 2 and other gases. Therefore, resource waste and environmental pollution are easily caused during the reuse process of soybean molasses.

[0003] Due to the rapid development of biosynthesis technology in recent years and the global energy shortage, especially molasses resources, which are the main carbon-based sources for microbial fermentation, are resources contended by many traditional fermentation industries and emerging biosynthesis enterprises. As a biological resource that can be reused, soybean molasses has gradually attracted attention.

[0004] Currently, there are many research reports on the fermentation of soybean molasses. Chinese patent document CN103614421A discloses the production of citric acid by fermenting soybean molasses with Aspergillus niger; Chinese patent document CN103614422A discloses the production of acetic acid by fermenting soybean molasses with Acetobacter and yeast; in addition, the prior art also reports the production of bacterial cellulose, alcohol by fermenting soybean molasses with Acetobacter xylinum, and the production of fructan by fermenting soybean molasses with Bacillus licheniformis, all of which have achieved certain effects, indicating that soybean molasses has important development value. In terms of feed, the prior art reports that the waste residue after fermenting soybean molasses to produce alcohol can be used as feed, but due to the large amount of raffinose, stachyose, and saponins in soybean molasses, it will affect animal production performance and feed digestibility. In addition, in terms of feed, it is required to use microbial strains permitted by the announcements of the Ministry of Agriculture and Rural Affairs of China. As a permitted strain, there is currently no research on using yeast alone to ferment soybean molasses and apply it to animal production, and there are also very few research reports on reducing raffinose, stachyose, and saponins in soybean molasses through yeast fermentation. Summary of the Invention

[0005] The object of the present invention is to provide a Saccharomyces cerevisiae capable of fermenting soybean molasses and its application. This Saccharomyces cerevisiae can use soybean molasses as the main fermentation sugar source, effectively utilize carbon-based substances such as sucrose, raffinose, and stachyose in soybean molasses, reduce antinutritional factors such as saponins in soybean molasses, and the yeast-related fermentation products obtained thereby can be directly added to livestock and poultry feeds.

[0006] To this end, in the first aspect, the present invention provides a Saccharomyces cerevisiae SC-HKB100, which was deposited at the China Center for Type Culture Collection on September 30, 2019, with the address: Wuhan University, Wuhan, China, and the deposit number is CCTCC NO: M 2019769.

[0007] In the second aspect of the present invention, an application of the Saccharomyces cerevisiae in fermenting soybean molasses is provided.

[0008] In the third aspect of the present invention, a preparation method for the Saccharomyces cerevisiae to produce fermentation products using soybean molasses is provided, which includes a liquid aerobic fermentation step. The liquid aerobic fermentation step includes: inoculating the seed liquid of the Saccharomyces cerevisiae into a soybean molasses liquid medium and performing aerobic fermentation to obtain a fermentation culture solution.

[0009] Further, the soybean molasses liquid medium is prepared by diluting soybean molasses with water by 2 - 3 times and adjusting the pH value to 5.8 - 6.8.

[0010] Further, in the liquid aerobic fermentation, the inoculation amount of the seed liquid is 18% - 25%, such as 18%, 20%, 25%, etc.; the fermentation temperature is 28°C - 32°C, the fermentation time is 24 - 36 h, and the ventilation volume is 1 - 4 L / min to obtain a fermentation culture solution.

[0011] Further, the preparation method further includes a seed liquid culture step before the liquid aerobic fermentation step; the seed liquid culture step includes: inoculating the Saccharomyces cerevisiae into a seed medium and performing seed culture to obtain the seed liquid of the yeast.

[0012] Further, the seed medium includes 145 g / L of malt extract powder and 0.08 g / L of chloramphenicol, and the pH value is 5.8 - 6.8.

[0013] Further, the temperature of the seed culture is 28 - 32°C, the rotation speed is 120 - 200 r / min, and the culture time is 24 - 36 h.

[0014] Further, the preparation method further includes a strain activation step before the seed liquid culture step; the strain activation step includes: inoculating the Saccharomyces cerevisiae into a slant medium and performing strain activation culture.

[0015] Further, the slant medium is a solid wort medium, and the solid wort medium includes malt extract powder, chloramphenicol, and agar; the content of each component is preferably 130 g / L of malt extract powder, 0.1 g / L of chloramphenicol, and 20 g / L of agar.

[0016] Further, the temperature for the activation culture of the strain is 28 - 32 °C, and the culture time is 24 - 48 h.

[0017] Further, the preparation method further includes a drying step after the liquid aerobic fermentation step, and the drying step includes: drying the fermentation culture solution to prepare yeast powder.

[0018] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0019] 1) The Saccharomyces cerevisiae SC-HKB100 provided by the present invention has strong fermentation ability, can fully utilize components such as sucrose, raffinose, and stachyose in soybean molasses, reduce the content of anti-nutritional factors such as saponins, and improve the yeast fermentation utilization rate of soybean molasses;

[0020] 2) The Saccharomyces cerevisiae SC-HKB100 provided by the present invention can efficiently utilize soybean molasses for fermentation compared with traditional molasses, and the obtained yeast quantity, solids content, and protein content are not lower than the fermentation effect of traditional molasses;

[0021] 3) The preparation method of the present invention can efficiently utilize soybean molasses for fermentation production, and can produce fermentation products such as yeast, yeast hydrolysate, yeast extract, and yeast culture, reduce the production cost of this series of products, and indirectly reduce the application cost of animals;

[0022] 4) The fermentation product obtained by the present invention using soybean molasses as a fermentation sugar source has a high protein content and rich amino acid nutrition, and can be used as a high-quality protein raw material;

[0023] 5) The soybean molasses selected as the raw material of the present invention has rich sources and low prices, not only reduces the production cost, but also reduces problems such as resource waste and environmental pollution caused by waste, thus achieving the purpose of maximizing resource utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] By reading the detailed description of the preferred embodiments below, various other advantages and benefits will become clear to those of ordinary skill in the art. The drawings are only for the purpose of showing the preferred embodiments and are not considered to be a limitation of the present invention. In the drawings:

[0025] Figure 1 : Growth of Saccharomyces cerevisiae SC-HKB100 in soybean molasses solid medium after 13 h of fermentation;

[0026] Figure 2 : Growth of Saccharomyces cerevisiae SC-HKB100 in solid medium after 16 h of fermentation; among them, Figure 2 (Left) is the soybean molasses solid medium, Figure 2 (Right) is the wort medium;

[0027] Figure 3 : Statistical results of the number of bacteria of Saccharomyces cerevisiae SC-HKB100 in soybean molasses medium and beet molasses medium with different mass percentages after 24 h of fermentation;

[0028] Figure 4 : Detection results of the soluble solid content of Saccharomyces cerevisiae SC-HKB100 in soybean molasses medium and beet molasses medium with different mass percentages after 24 h of fermentation;

[0029] Figure 5 : Detection results of the protein OD value of Saccharomyces cerevisiae SC-HKB100 in soybean molasses medium and beet molasses medium with different mass percentages after 24 h of fermentation. Detailed implementation manners

[0030] The exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. Although the exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided so that the present disclosure can be more thoroughly understood and the scope of the present disclosure can be fully communicated to those skilled in the art.

[0031] Example 1 Isolation of Saccharomyces cerevisiae

[0032] (1) Medium: Wort medium. Take 500 g of malt, crush it and put it into a beaker, add 2000 mL of distilled water, stir and heat in a water bath at 45 °C for 30 min, then raise the temperature to 70 °C and keep it for 1 h; filter with 8 layers of gauze, and wash the beaker and filter tank with distilled water to make the filtrate reach 2000 mL, boil it and then filter again, cool it, and store it at 10 °C for later use.

[0033] (2) Isolation of yeast: Using beer, wine, pickles, fermented fruit and vegetable juices, dough, koumiss, etc. as raw materials, collect 400 mL (liquid sample) or 400 g (mashed solid sample) of raw materials respectively, place them in a sterile conical flask, cover with 8 layers of gauze and ferment naturally at 28 °C for 3 d. Under sterile conditions, shake the fermented culture solution sample well, and use a sterile pipette to suck an appropriate amount of the fermented liquid into the sterilized wort culture medium, and culture at 28 °C. When there is a wine smell, use the gradient dilution method to dilute to a concentration of 10 -4, 0.1 mL of the diluted culture solution was taken and evenly spread on the malt extract medium (malt extract culture solution + 2% agar). It was inverted at 28 °C for 48 h. After colonies grew, those with typical single colonies (yeast colonies) were selected and further purified by streaking 3 times. After being examined under a microscope as pure cultures, they were transferred onto malt extract solid slant media respectively and stored at 4 °C for later use.

[0034] Cultivation was carried out using the malt extract medium, and the growth situation during the observation period was observed. The strains with the highest survival rate and the most vigorous growth were selected for the next test of degrading raffinose oligosaccharides in soybean molasses. A total of 8 strains of yeast were obtained, namely: HKB-6, HKB-10, HKB-33, HKB-36, SC-HKB60, SC-HKB75, SC-HKB100, and SC-HKB105.

[0035] Example 2 Screening of Saccharomyces cerevisiae

[0036] (1) Fermentation of soybean molasses: Under aseptic conditions, 2 loops were taken from the malt extract solid slant media of the 8 strains of Saccharomyces cerevisiae screened in Example 1 and stored for later use with an inoculation loop respectively, and were inoculated into soybean molasses (without other components). The pH of the soybean molasses medium was adjusted to 6.2, and fermentation was carried out at a fermentation temperature of 28 °C for 36 h. At the same time, a blank control group without inoculating yeast was set up.

[0037] (2) Determine the contents of raffinose, stachyose, and saponins in unfermented soybean molasses and soybean molasses fermented by yeast. The determination results are shown in Table 1:

[0038] Table 1 Effects of fermentation by different yeasts on the contents of raffinose, stachyose, and saponins in soybean molasses

[0039] Group Raffinose (%) Stachyose (%) Saponin (%) Unfermented group 2.60 9.01 1.08 Blank control group 2.59 8.97 1.07 HKB-6 1.50 7.01 0.76 HKB-10 2.61 8.94 1.09 HKB-3 2.55 9.01 1.07 HKB-36 2.58 8.78 1.02 SC-HKB60 2.58 8.89 1.04 SC-HKB75 2.58 8.95 1.07 SC-HKB100 0.35 1.65 0.12 SC-HKB105 1.04 4.02 0.43

[0040] As can be seen from Table 1, soybean molasses contains relatively high contents of raffinose, stachyose, and saponins. If yeast is not inoculated and only treated under fermentation conditions, the contents of raffinose, stachyose, and saponins in it do not change significantly (blank control group). Among the 8 strains of Saccharomyces cerevisiae, 5 strains, namely HKB-10, HKB-33, HKB-36, SC-HKB60, and SC-HKB75, cannot degrade the oligosaccharides in soybean molasses; the remaining HKB-6, SC-HKB100, and SC-HKB105 can all degrade raffinose, stachyose, and saponins in soybean molasses to varying degrees, and among them, SC-HKB100 has the best degradation effect on raffinose, stachyose, and saponins in soybean molasses, and the degradation rates reach 86.54%, 81.69%, and 88.89% respectively.

[0041] Therefore, Saccharomyces cerevisiae SC-HKB100 was selected as the fermentation strain for fermenting soybean molasses.

[0042] Growth in the Soybean Molasses Medium in Example 3

[0043] (1) Solid Medium: The malt extract solid medium and the soybean molasses solid medium were prepared separately, and Saccharomyces cerevisiae SC-HKB100 was inoculated into each of them and cultured at 28 °C and pH 6.2. The growth of colonies was observed during the culture process. According to the observation, after 13 h of culture, as Figure 1 shown, obvious colonies had grown on the soybean molasses medium, indicating that the soybean molasses as a single medium could fully meet the nutritional requirements of Saccharomyces cerevisiae SC-HKB100. After 16 h of culture, Figure 2 the colony growth in the soybean molasses medium ( Figure 2 left) and the malt extract medium ( Figure 2 right) are shown respectively. It can be seen that compared with the commonly used high-nutrient malt extract medium, the use of soybean molasses for the culture of Saccharomyces cerevisiae SC-HKB100 had no negative impact on its growth, indicating that the bacterium had a very high utilization rate of soybean molasses.

[0044] (2) Liquid Medium: Saccharomyces cerevisiae SC-HKB100 was inoculated into the malt extract liquid medium and the soybean molasses liquid medium at an inoculation amount of 1% respectively, and shake-flask fermentation culture was carried out under the same conditions; the growth of the bacterial cells was detected at 12 h and 16 h of culture respectively. The detection results showed that there was no obvious difference between the soybean molasses liquid medium and the malt extract liquid medium. After 12 h of culture, the number of bacteria was about 8.4×10 11 , and after 16 h of culture, the number of bacteria was about 2.2×10 12 .

[0045] Example 4 Influence of the Inoculation Amount of the Seed Liquid on the Fermentation Effect of Soybean Molasses

[0046] (1) Activation of the Slant Culture: The cryopreserved Saccharomyces cerevisiae SC-HKB100 was inoculated onto a slant medium containing 130 g / L of malt extract powder, 0.1 g / L of chloramphenicol, and 20 g / L of agar, and incubated at a constant temperature of 30 °C for 48 h to obtain the activated yeast culture.

[0047] (2) Preparation of the Seed Liquid: The activated yeast culture was inoculated with 1 loop into a liquid medium containing 145 g / L of malt extract powder, 0.08 g / L of chloramphenicol, and a pH value of 6.0, and cultured in a constant-temperature shaking incubator at 28 °C with a rotation speed of 200 r / min for 24 h.

[0048] (3) Fermentation of soybean molasses: The prepared seed liquid was inoculated into the diluted soybean molasses medium at inoculation amounts of 10%, 20%, and 30% respectively. The dilution factor of soybean molasses was 2 times, that is, water was added to dilute soybean molasses to 2 times its mass. The pH value was 6.2, and then enlarged culture was carried out. The aeration rate was controlled at 4 L / min, the temperature was 28 °C, and the fermentation time was 36 h to obtain the fermentation product.

[0049] (4) Determine the contents of raffinose, stachyose, and saponins in the fermentation products with different inoculation amounts (the contents were converted into mass fractions based on soybean molasses). The determination results are shown in Table 2:

[0050] Table 2 Influence of seed liquid inoculation amount on the fermentation effect of soybean molasses

[0051] Inoculum size Raffinose (%) Stachyose (%) Saponin (%) 10％ 0.39 1.59 0.14 20％ 0.30 1.57 0.10 30％ 0.34 1.64 0.12

[0052] It can be seen from Table 2 that the contents of raffinose, stachyose, and saponins in the products after fermenting soybean molasses with different inoculation amounts were significantly reduced, and the degradation rates all reached more than 80%. The differences in fermentation effects were not very significant, but the fermentation effect was the best at an inoculation amount of 20%.

[0053] Example 5 Comparison between fermentation of soybean molasses and traditional molasses fermentation

[0054] (1) Activation of slant strains: The cryopreserved Saccharomyces cerevisiae SC-HKB100 was inoculated onto a slant medium containing 130 g / L of malt extract powder, 0.1 g / L of chloramphenicol, and 20 g / L of agar, and left standing at a constant temperature of 28 °C for 24 h to obtain the activated yeast strains.

[0055] (2) Preparation of seed liquid: The activated yeast strains were inoculated with 1 loop into a liquid medium containing 145 g / L of malt leachate powder, 0.08 g / L of chloramphenicol, and a pH value of 5.8, and cultured in a constant temperature shaker at 32 °C with a rotation speed of 120 r / min for 36 h.

[0056] (3) Fermentation comparison: The prepared seed liquid was inoculated at an inoculation amount of 20% into soybean molasses media and beet molasses media with different mass ratios (6%, 8%, 10%, 12%, and 15%) respectively. The pH value was 6.2, the aeration rate was controlled at 1 L / min, the temperature was 32 °C, and the fermentation time was 24 h.

[0057] (4) Comparison of fermentation results: After the fermentation was completed, the fermentation products were detected, and the detection results are shown in Figures 3 - 5As shown. According to the detection results, Saccharomyces cerevisiae SC-HKB100 can utilize soybean molasses well, obtaining a yeast count similar to that of traditional fermentation sugar source beet molasses, and the contents of solids, protein, etc. are significantly higher than those of beet molasses fermentation, indicating that this yeast has excellent industrial application value.

[0058] Example 6 Analysis of Saccharomyces cerevisiae SC-HKB100 Fermented with Soybean Molasses

[0059] After spray-drying the Saccharomyces cerevisiae SC-HKB100 produced in the soybean molasses medium, a comparative analysis was carried out with the protein content and amino acids in soybean meal, and the results are shown in Table 3.

[0060] Table 3 Comparison Results of Yeast Powder Protein Prepared by Saccharomyces cerevisiae SC-HKB100 Using Soybean Molasses Fermentation and Soybean Protein

[0061]

[0062]

[0063] According to the detection results in Table 3, in the fermentation product of Saccharomyces cerevisiae SC-HKB100 provided by the present invention using soybean molasses, the protein content is 52%, the proportion of essential amino acids in total amino acids (EAA / TAA) is 47.58%, and the ratio of essential amino acids to non-essential amino acids (EAA / NEAA) is 0.90. According to the EAA / TAA value (40%) and EAA / NEAA value (0.6) stipulated by the FAO / WHO standard, the fermentation product of Saccharomyces cerevisiae provided by the present invention using soybean molasses belongs to high-quality protein, and its protein amino acid value is higher than that of soybean meal and can be used as a high-quality protein raw material.

[0064] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A Saccharomyces cerevisiae SC-HKB100, which was deposited with the China Center for Type Culture Collection on September 30, 2019, and the deposit number is CCTCC NO: M 2019769.

2. Use of the Saccharomyces cerevisiae according to claim 1 in fermenting soybean molasses.

3. A preparation method for a Saccharomyces cerevisiae to produce fermentation products using soybean molasses, characterized in that, it includes a liquid aerobic fermentation step, and the liquid aerobic fermentation step includes: inoculating the seed liquid of the Saccharomyces cerevisiae according to claim 1 into a soybean molasses liquid medium, and performing aerobic fermentation to obtain a fermentation culture solution.

4. The preparation method according to claim 3, characterized in that, the soybean molasses liquid medium is prepared by diluting soybean molasses with water by 2 - 3 times and adjusting the pH value to 5.8 - 6.

8.

5. The preparation method according to claim 3, characterized in that, in the liquid aerobic fermentation, the inoculation amount of the seed liquid is 18% - 25%, the fermentation temperature is 28°C - 32°C, the fermentation time is 24 - 36 h, and the ventilation volume is 1 - 4 L / min to obtain a fermentation culture solution.

6. The preparation method according to claim 3, characterized in that, the preparation method further includes a seed liquid culture step before the liquid aerobic fermentation step; the seed liquid culture step includes: inoculating the Saccharomyces cerevisiae into a seed medium and performing seed culture to obtain the seed liquid of the yeast.

7. The preparation method according to claim 6, characterized in that, the seed medium includes 145 g / L of malt extract powder, 0.08 g / L of chloramphenicol, and the pH value is 5.8 - 6.

8.

8. The preparation method according to claim 6, characterized in that, the temperature of the seed culture is 28 - 32°C, the rotation speed is 120 - 200 r / min, and the culture time is 24 - 36 h.

9. The preparation method according to claim 6, characterized in that, the preparation method further includes a strain activation step before the seed liquid culture step; the strain activation step includes: inoculating the Saccharomyces cerevisiae into a slant medium and performing strain activation culture.

10. The preparation method according to claim 9, characterized in that, the slant medium is a solid malt extract medium, and the solid malt extract medium includes malt extract powder, chloramphenicol, and agar.

11. The preparation method according to claim 9, characterized in that, the temperature of the strain activation culture is 28 - 32°C, and the culture time is 24 - 48 h.

12. The preparation method according to claim 3, characterized in that, the preparation method further includes a drying step after the liquid aerobic fermentation step, and the drying step includes: performing drying treatment on the fermentation culture solution.

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