CN116144515A

CN116144515A - Saccharomyces cerevisiae capable of fermenting by using soybean molasses and application thereof

Info

Publication number: CN116144515A
Application number: CN202211088219.2A
Authority: CN
Inventors: 刘明; 董彦君; 夏冰; 孙守志; 王文欢; 方洛云; 潘予琮
Original assignee: Beijing Zhongnong Hongke Biotechnology Co ltd; Beijing University of Agriculture
Current assignee: Beijing Zhongnong Hongke Biotechnology Co ltd; Beijing University of Agriculture
Priority date: 2022-09-07
Filing date: 2022-09-07
Publication date: 2023-05-23

Abstract

The invention relates to a saccharomyces cerevisiae capable of fermenting by using soybean molasses and application thereof, wherein the saccharomyces cerevisiae (Saccharomyces cerevisiae) SC-HKB100 is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of NO: m2019769. The saccharomyces cerevisiae SC-HKB100 can be fully fermented by taking the soybean molasses as a sugar source under specific conditions, so that sucrose, raffinose, stachyose and the like in the soybean molasses are effectively utilized, and anti-nutritional factors such as saponin and the like in the soybean molasses are reduced. Compared with the traditional beet molasses, cane molasses and other fermentation sugar sources, the production cost of the yeast fermentation industry can be obviously reduced, and the product with high protein content and rich amino acid nutrition can be obtained by fermentation, thus being used as an ideal high-quality protein raw material.

Description

Saccharomyces cerevisiae capable of fermenting by using soybean molasses and application thereof

Technical Field

The invention relates to the technical field of fungi, in particular to saccharomyces cerevisiae capable of fermenting by using soybean molasses and application thereof.

Background

Soy molasses is a byproduct of production of soy protein concentrate, is brownish red and has intense sweetness, and its main components are sugar (soybean oligosaccharide), protein, phospholipid, ash, saponins, isoflavone, organic acid and other phytochemicals. The soy molasses is widely available and inexpensive, and it is reported that greater than 0.34 tons of soy molasses will be produced per 1 ton of soy protein concentrate produced by the alcohol process. However, due to the presence of oligosaccharides (such as raffinose, stachyose, etc.), saponins, etc. in the soy molasses, conventional industrial yeast, etc. microorganisms cannot fully utilize their sugar content; if added directly into livestock and fowl feed, a large amount of oligosaccharide can not be digested in animal digestive tract, and CH is produced by microbial fermentation decomposition ₄ 、H ₂ 、CO ₂ And the gases are the same, so that resource waste and environmental pollution are easily caused in the recycling process of the soybean molasses.

Due to the rapid development of biosynthesis technology in recent years, global energy is lacking, and especially molasses resources, which are the main carbon-based source for microbial fermentation, are the resources contending for many traditional fermentation industries and emerging biosynthesis enterprises. Soy molasses is becoming a biological resource that can be reused.

At present, many research reports are about the fermentation of soy molasses, and China patent document CN103614421A discloses the use of Aspergillus niger to ferment the soy molasses for producing citric acid; chinese patent document CN103614422a discloses the production of acetic acid by fermentation of soy molasses using acetobacter and yeast; in addition, the prior art reports that bacterial cellulose is produced by fermenting the soybean molasses by using acetobacter xylinum, alcohol is produced, and the soybean molasses is fermented by using bacillus licheniformis to produce the levan and the like, so that a certain effect is obtained, and the soybean molasses has important development value. In the aspect of feed, the prior art reports that waste residues generated after the fermentation of the soybean molasses to produce alcohol can be used as feed, but the animal productivity and the feed digestibility are affected due to the fact that the soybean molasses contains a large amount of raffinose, stachyose, saponins and the like. In addition, in terms of feeds, the use of microbial species allowed for use in the national agricultural rural publications is required. Yeast is used as a permitted strain, and there is no research on fermenting soybean molasses solely by using it for animal production, and there is little research report on reducing raffinose, stachyose and saponin in soybean molasses by yeast fermentation.

Disclosure of Invention

The invention aims to provide a saccharomyces cerevisiae capable of fermenting by using soybean molasses and application thereof, the saccharomyces cerevisiae can take the soybean molasses as a main fermentation sugar source, carbon bases such as sucrose, raffinose, stachyose and the like in the soybean molasses can be effectively utilized, and anti-nutritional factors such as saponins and the like in the soybean molasses are reduced, so that the obtained yeast related fermentation product can be directly added into livestock and poultry feeds.

To this end, in a first aspect, the invention provides a Saccharomyces cerevisiae (Saccharomyces cerevisiae) SC-HKB100 deposited at the China center for type culture Collection, address: the preservation number of the Chinese university of Wuhan is CCTCC NO: m2019769.

In a second aspect, the invention provides the use of said Saccharomyces cerevisiae for fermenting soy molasses.

In a third aspect of the present invention, there is provided a process for producing a fermentation product by using soy molasses by the Saccharomyces cerevisiae, comprising a liquid aerobic fermentation step comprising: inoculating the seed liquid of the saccharomyces cerevisiae into a soybean molasses liquid culture medium, and performing aerobic fermentation to prepare a fermentation culture liquid.

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.

Further, in the liquid aerobic fermentation, the seed liquid is inoculated in an amount of 18% -25%, for example, 18%, 20%, 25%, etc.; the fermentation temperature is 28-32 ℃, the fermentation time is 24-36h, and the ventilation is 1-4L/min, thus obtaining the fermentation culture solution.

Further, the preparation method further comprises a seed liquid culture step positioned before the liquid aerobic fermentation step; the seed liquid culture step comprises the following steps: inoculating the saccharomyces cerevisiae into a seed culture medium, and carrying out seed culture to obtain a seed solution of the saccharomyces cerevisiae.

Further, the seed culture medium comprises 145g/L of malt extract powder, 0.08g/L of chloramphenicol and a pH value of 5.8-6.8.

Further, the temperature of the seed culture is 28-32 ℃, the rotating speed is 120-200r/min, and the culture time is 24-36h.

Further, the preparation method further comprises a strain activation step positioned before the seed liquid culture step; the strain activation step comprises the following steps: inoculating the saccharomyces cerevisiae into a slant culture medium, and performing strain activation culture.

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

Further, the temperature of the strain activation culture is 28-32 ℃, and the culture time is 24-48 hours.

Further, the preparation method further comprises a drying step after the liquid aerobic fermentation step, the drying step comprising: and drying the fermentation culture solution to obtain the yeast powder.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

1) The saccharomyces cerevisiae SC-HKB100 provided by the invention has strong fermentation capability, can fully utilize the components such as sucrose, raffinose, stachyose and the like in the soybean molasses, reduce the content of anti-nutritional factors such as saponin and the like, and improve the yeast fermentation utilization rate of the soybean molasses;

2) Compared with the traditional molasses, the saccharomyces cerevisiae SC-HKB100 provided by the invention can efficiently utilize the soybean molasses for fermentation to obtain the fermentation effect that the quantity of yeast, the solid content and the protein content are not lower than those of the traditional molasses;

3) The preparation method can efficiently utilize the soybean molasses for fermentation production, can produce fermentation products such as saccharomycetes, yeast hydrolysate, yeast extract, yeast culture and the like, reduces the production cost of the series of products, and indirectly reduces the application cost of animals;

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

5) The soybean molasses has rich sources of raw materials and low price, not only reduces the production cost, but also reduces the problems of resource waste, environmental pollution and the like caused by waste, thereby realizing the purpose of maximizing the resource utilization.

Drawings

Various other advantages and benefits will become apparent to those of ordinary skill in the art upon reading the following detailed description of the preferred embodiments. The drawings are only for purposes of illustrating the preferred embodiments and are not to be construed as limiting the invention. In the drawings:

fig. 1: the growth condition of saccharomyces cerevisiae SC-HKB100 in a soybean molasses solid medium for 13 hours;

fig. 2: the growth condition of saccharomyces cerevisiae SC-HKB100 in a solid culture medium for 16 hours; wherein, FIG. 2 (left) is a solid medium of soy molasses and FIG. 2 (right) is a medium of wort;

fig. 3: the bacterial count statistical result of Saccharomyces cerevisiae SC-HKB100 fermented for 24 hours in soybean molasses culture mediums and beet molasses culture mediums with different mass percentages;

fig. 4: the detection result of the soluble solid content of Saccharomyces cerevisiae SC-HKB100 in soybean molasses culture medium and beet molasses culture medium with different mass percentages for 24 hours;

fig. 5: results of protein OD value detection of Saccharomyces cerevisiae SC-HKB100 fermented in different mass percentages of the soybean molasses medium and beet molasses medium for 24 hours.

Detailed Description

Exemplary embodiments of the present disclosure will be described in more detail below with reference to the accompanying drawings. While exemplary embodiments of the present disclosure are shown in the drawings, it should be understood that the present disclosure may be embodied in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the disclosure to those skilled in the art.

EXAMPLE 1 isolation of Saccharomyces cerevisiae

(1) Culture medium: wort medium. Taking 500g of malt, crushing the malt, putting the crushed malt into a beaker, adding 2000mL of distilled water, stirring the mixture in a water bath at 45 ℃ for 30min, adjusting the temperature to 70 ℃, and keeping the temperature for 1h; the 8 layers of gauze are filtered, the beaker and the filter tank are washed by using a positive Liu Shu, the filtrate reaches 2000mL, and after boiling, the filtrate is cooled and stored at 10 ℃ for standby.

(2) Isolation of yeasts: taking beer, wine, pickle, fermented fruit and vegetable juice, dough, mare's milk, etc. as raw materials, respectively collecting 400mL (liquid sample) or 400g (mashed solid sample) raw materials, placing into a sterile conical flask, covering 8 layers of gauze, and naturally fermenting at 28deg.C for 3d. Under aseptic condition, shake the fermentation broth sample evenly, draw proper amount of fermentation broth into sterilized wort broth by aseptic pipettor, and culture at 28deg.C. Diluting with gradient dilution method until the concentration is 10 ^-4 Sucking 0.1mL of diluted culture solution, uniformly coating on a wort culture medium (wort culture solution+2% agar), inverting at 28 ℃ for 48 hours, selecting a typical single colony (saccharomycete colony) after bacterial colony grows out, further streaking and purifying for 3 times, performing microscopic examination to obtain pure cultures, respectively transferring to a wort solid slant culture medium, and preserving at 4 ℃ for later use.

Culturing with wort culture medium, observing growth condition, and selecting bacteria with highest survival rate and most luxuriant growth for the next test of degrading soybean molasses oligosaccharide. 8 strains of saccharomycetes are obtained, and the saccharomycetes are respectively as follows: HKB-6, HKB-10, HKB-33, HKB-36, SC-HKB60, SC-HKB75, SC-HKB100 and SC-HKB105.

EXAMPLE 2 screening of Saccharomyces cerevisiae

(1) Fermenting soybean molasses: under the aseptic condition, 2 loops are respectively taken from the wort solid slant culture medium for preserving 8 strains of saccharomyces cerevisiae obtained by screening in the example 1 by using an inoculating loop, the wort solid slant culture medium is respectively inoculated into soybean molasses (without other components), the pH of the soybean molasses culture medium is regulated to 6.2, and the fermentation is carried out for 36 hours at the fermentation temperature of 28 ℃. A blank control group without yeast inoculation was also set.

(2) The raffinose, stachyose and saponin contents of the unfermented and yeast-fermented soy molasses were determined. The measurement results are shown in Table 1:

TABLE 1 Effect of different Yeast fermentation on the content of raffinose, stachyose and saponins in soy molasses

Group of	Raffinose (%)	Stachyose (%)	Saponins (%)
				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

As can be seen from Table 1, the soy molasses contains higher levels of raffinose, stachyose and saponins. If yeast is not inoculated, the fermentation conditions are adopted only for treatment, and the content of raffinose, stachyose and saponin in the fermentation broth is not changed obviously (blank control). Among 8 strains of Saccharomyces cerevisiae, 5 strains of Saccharomyces cerevisiae, HKB-10, HKB-33, HKB-36, SC-HKB60 and SC-HKB75, cannot degrade oligosaccharides in the soybean molasses; the residual HKB-6, SC-HKB100 and SC-HKB105 can degrade raffinose, stachyose and saponin in the soy molasses to different degrees, and the SC-HKB100 has the best degradation effect on the raffinose, stachyose and saponin in the soy molasses, and the degradation rates respectively reach 86.54%, 81.69% and 88.89%.

Thus, saccharomyces cerevisiae SC-HKB100 was selected as the fermentation broth for fermentation using soy molasses.

Example 3 growth Using Soy molasses media

(1) Solid medium: respectively preparing a wort solid culture medium and a soybean molasses solid culture medium, respectively inoculating Saccharomyces cerevisiae SC-HKB100, culturing at 28 ℃ and pH6.2, and observing colony growth in the culture process. According to the observation, after 13h of cultivation, as shown in FIG. 1, it can be observed that obvious colonies have grown on the soy molasses medium, indicating that the nutritional requirements of Saccharomyces cerevisiae SC-HKB100 can be fully satisfied with the soy molasses as a single medium. After 16h of incubation, FIG. 2 shows colony growth using soy molasses medium (left of FIG. 2) and wort medium (right of FIG. 2), respectively. It can be seen that the cultivation of Saccharomyces cerevisiae SC-HKB100 with soy molasses has no negative effect on its growth compared to the usual high nutrient medium, wort medium, indicating that the use of this strain for soy molasses is very high.

(2) Liquid medium: inoculating Saccharomyces cerevisiae SC-HKB100 to a malt juice liquid medium and a soybean molasses liquid medium respectively according to an inoculum size of 1%, and carrying out shake flask fermentation culture under the same conditions; the growth of the bacterial cells is detected respectively at 12h and 16h, and the detection result shows that the liquid culture medium of the soybean molasses and the liquid culture medium of the malt juice are not obviously different, and the bacterial count is about 8.4x10 after 12h of culture ¹¹ After 16 hours of cultivation, the bacterial count was about 2.2X10 ¹² 。

Example 4 Effect of seed liquid inoculum size on fermentation Effect of Soybean molasses

(1) Activating inclined plane strains: inoculating the frozen and preserved saccharomyces cerevisiae SC-HKB100 to a slant culture medium containing 130g/L malt extract powder, 0.1g/L chloramphenicol and 20g/L agar, standing at a constant temperature of 30 ℃, and culturing for 48 hours to obtain the activated saccharomyces cerevisiae.

(2) Preparing seed liquid: inoculating the activated yeast strain into a liquid culture medium containing 145g/L malt extract powder, 0.08g/L chloramphenicol and 6.0 pH value by using 1 loop, and culturing at a constant temperature of 28 ℃ for 24h at a rotating speed of 200 r/min.

(3) Fermenting soybean molasses: inoculating the prepared seed solution into diluted soy molasses culture medium with 10%, 20% and 30% inoculum size respectively, wherein the dilution factor of the soy molasses is 2 times, namely: adding water to dilute the soybean molasses to 2 times of the mass of the soybean molasses, performing expansion culture at pH of 6.2, controlling ventilation amount to 4L/min, and fermenting at 28deg.C for 36 hr to obtain fermentation product.

(4) The contents of raffinose, stachyose and saponins (the contents are converted into the mass parts of the soybean molasses) in the fermentation products with different inoculum sizes are measured. The measurement results are shown in Table 2:

TABLE 2 Effect of seed liquid inoculum size on fermentation effects of Soybean molasses

Inoculum size	Raffinose (%)	Stachyose (%)	Saponins (%)
				10％	0.39	1.59	0.14
20％	0.30	1.57	0.10
				30％	0.34	1.64	0.12

As shown in Table 2, the seed sugar, stachyose and saponin contents of the products obtained by fermenting the soy molasses with different inoculum sizes are all significantly reduced, the degradation rate is up to 80% or more, the fermentation effect difference is not very significant, but the fermentation effect is optimal with 20% inoculum size.

Example 5 comparison of soy molasses fermentation and conventional molasses fermentation

(1) Activating inclined plane strains: inoculating the frozen and preserved saccharomyces cerevisiae SC-HKB100 to a slant culture medium containing 130g/L malt extract powder, 0.1g/L chloramphenicol and 20g/L agar, standing at the constant temperature of 28 ℃, and culturing for 24 hours to obtain activated yeast strains.

(2) Preparing seed liquid: inoculating the activated yeast strain into a liquid culture medium containing 145g/L malt extract powder, 0.08g/L chloramphenicol and pH value of 5.8 by using 1 loop, and culturing at a constant temperature of 32deg.C at a rotation speed of 120r/min; culturing for 36h.

(3) Fermentation comparison: the prepared seed liquid is inoculated into soybean molasses culture medium and beet molasses culture medium with different mass ratios (6%, 8%, 10%, 12% and 15%) respectively at 20% inoculum size, pH value is 6.2, aeration rate is controlled to be 1L/min, temperature is 32 ℃, and fermentation time is 24h.

(4) And (3) comparing fermentation results: after the fermentation is finished, the fermentation product is detected, and the detection result is shown in figures 3-5. According to the detection result, the Saccharomyces cerevisiae SC-HKB100 can well utilize the soybean molasses to obtain the quantity of yeasts similar to that of the traditional fermented sugar beet molasses, and the contents of solids, proteins and the like are obviously higher than the fermentation effect of the beet molasses, so that the saccharomycetes have good industrial application value.

EXAMPLE 6 analysis of Saccharomyces cerevisiae SC-HKB100 fermented with soy molasses

The Saccharomyces cerevisiae SC-HKB100 produced by the soybean molasses culture medium is subjected to spray drying, and then is subjected to comparison analysis with the protein content and amino acid in the soybean meal, and the results are shown in Table 3.

TABLE 3 comparison of Yeast powder protein and Soybean protein by Saccharomyces cerevisiae SC-HKB100 prepared by fermentation of Soybean molasses

According to the detection results in Table 3, the Saccharomyces cerevisiae SC-HKB100 provided by the invention utilizes the fermentation product of the soybean molasses, the protein content is 52%, the ratio of essential amino acids to 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 the EAA/NEAA value (0.6) specified by the FAO/WHO standard, the product of the Saccharomyces cerevisiae fermentation soybean molasses provided by the invention belongs to high-quality protein, and the protein amino acid value of the product is higher than that of soybean meal, so that the product can be used as a high-quality protein raw material.

The present invention is not limited to the above-mentioned embodiments, and any changes or substitutions that can be easily understood by those skilled in the art within the technical scope of the present invention are intended to be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope of the claims.

Claims

1. Saccharomyces cerevisiae (Saccharomyces cerevisiae) SC-HKB100 was deposited at China center for type culture Collection (CCTCC NO) at 9.30.2019: m2019769.

2. Use of saccharomyces cerevisiae according to claim 1 for fermenting soy molasses.

3. A method for preparing a fermentation product by using soy molasses by saccharomyces cerevisiae, which is characterized by comprising a liquid aerobic fermentation step, wherein the liquid aerobic fermentation step comprises the following steps: inoculating the saccharomyces cerevisiae seed solution in the soy molasses liquid culture medium, and performing aerobic fermentation to obtain a fermentation culture solution.

4. The process according to claim 3, wherein the soybean molasses liquid medium is prepared by diluting soybean molasses with water 2 to 3 times and adjusting pH to 5.8 to 6.8.

5. The method according to claim 3, wherein in the aerobic fermentation of the liquid, the seed liquid is inoculated in an amount of 18% -25%, the fermentation temperature is 28 ℃ -32 ℃, the fermentation time is 24-36h, and the aeration amount is 1-4L/min, so as to obtain a fermentation culture liquid.

6. The method of claim 3, further comprising a seed liquid culturing step prior to the liquid aerobic fermentation step; the seed liquid culture step comprises the following steps: inoculating the saccharomyces cerevisiae into a seed culture medium, and carrying out seed culture to obtain a seed solution of the saccharomyces cerevisiae.

7. The method of claim 6, wherein the seed medium comprises malt extract powder 145g/L, chloramphenicol 0.08g/L, and pH 5.8-6.8;

preferably, the temperature of the seed culture is 28-32 ℃, the rotating speed is 120-200r/min, and the culture time is 24-36h.

8. The method according to claim 6, further comprising a seed-culture activating step before the seed-liquid culturing step; the strain activation step comprises the following steps: inoculating the saccharomyces cerevisiae into a slant culture medium, and performing strain activation culture.

9. The method of claim 8, wherein the slant medium is a solid wort medium comprising malt extract powder, chloramphenicol and agar;

preferably, the temperature of the strain activation culture is 28-32 ℃, and the culture time is 24-48h.

10. A method of preparing according to claim 3, further comprising a drying step following the liquid aerobic fermentation step, the drying step comprising: and drying the fermentation culture solution.