CN115074261A - Saccharomyces boulardii, fermentation medium thereof, ruminant feed and preparation method thereof - Google Patents
Saccharomyces boulardii, fermentation medium thereof, ruminant feed and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a saccharomyces boulardii and a fermentation medium thereof, a ruminant feed and a preparation method thereof, and belongs to the technical field of fermentation. The Latin yeast of the invention is named as Saccharomyces boulardii, is preserved in the common microorganism center of China Committee for culture Collection of microorganisms, has the address of No. 3 of No. 1 Xinchen Xilu in the area of the rising of the south of Beijing, the preservation date of 2022 years, 4 months and 21 days, and the preservation number of CGMCC No. 24750. The strain can be used for fully utilizing the waste lees and the bottom boiler water to prepare the ruminant feed.
Description
Technical Field
The invention relates to the technical field of fermentation, in particular to saccharomyces boulardii and a fermentation medium thereof, a ruminant feed and a preparation method thereof.
Background
Solid brewing is the main mode of liquor production at present, main byproducts of the solid brewing comprise waste lees, yellow water, bottom boiler water and the like, and high-valued recycling comprehensive utilization of the byproducts is particularly important while the liquor production capacity is continuously increased.
The waste lees are one of main byproducts in the production process of white spirit, and according to statistics, 6-10 tons of waste lees are generated when 1 ton of white spirit is produced. The resource utilization of the waste lees mainly aims at the conversion, degradation and reutilization of residual starch, protein, wood fiber and the like in the waste lees, such as the production of waste lees yeast, feed, culture medium, organic fertilizer, seasoning, fuel rod, white carbon black and the like. For the application of the solid white spirit waste lees as feed, small enterprises generally do not reprocess the solid white spirit waste lees, but directly provide the waste lees for surrounding farmers, but the solid white spirit waste lees are low in price, generally 80-100 yuan/ton. Such a treatment method has two disadvantages: on one hand, because the fresh spent grains are not processed, the domestic animals have difficulty in digesting and absorbing the fresh spent grains, so that the waste of resources is caused; on the other hand, the problem of disposal and utilization of a large amount of waste lees in large-scale white spirit brewing enterprises cannot be fundamentally solved because the waste lees have high moisture content, are easy to mildew and are difficult to convey.
The bottom boiler water is another byproduct in the production of white spirit, and refers to water remained at the bottom after the mixed grains of fermented grains are screened and distilled. At present, the bottom boiler water has no other place except entering a wastewater treatment system, and related comprehensive utilization research is blank.
Effectively utilizes the waste lees and the bottom boiler water, not only can reduce the cost of wastewater treatment in the white spirit industry, but also can provide a high-valued comprehensive utilization approach for the byproduct waste lees in the white spirit industry. However, the prior art does not disclose how to fully utilize the waste lees and the bottom boiler water to prepare the feed.
Disclosure of Invention
The invention aims to provide a saccharomyces boulardii and a fermentation medium thereof, a ruminant feed and a preparation method thereof.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a Saccharomyces boulardii, which is preserved in China general microbiological culture Collection center with the Latin name of Saccharomyces boulardii, the address of No. 3 Xilu-Chen-Yang No. 1 Hospital, No. 3, the preservation date of No. 2022, 4 and 21 days, and the preservation number of CGMCC No. 24750.
The invention also provides a composite bacterium containing the saccharomyces boulardii, which also comprises lactobacillus paracasei and lactobacillus buchneri;
the initial viable bacteria concentration of the saccharomyces boulardii in the composite bacteria is 1.0-2.0 multiplied by 10 8 cfu/mL；
The initial viable bacteria concentration of the lactobacillus paracasei in the composite bacteria is 0.5-1.0 multiplied by 10 8 cfu/mL；
The initial viable bacteria concentration of the lactobacillus buchneri in the composite bacteria is 1-3 multiplied by 10 7 cfu/mL。
The invention also provides a fermentation medium of the saccharomyces boulardii or the compound bacteria, and the fermentation medium comprises the following components:
waste lees, cottonseed meal, soybean meal, bran and pot water.
Preferably, the mass-volume ratio of the waste lees to the bottom boiler water is 35-39 g: 44-48 mL;
the mass volume ratio of the cottonseed meal to the bottom boiler water is 24-28 g: 44-48 mL;
the mass volume ratio of the soybean meal to the bottom boiler water is 20-24 g: 44-48 mL;
the mass volume ratio of the bran to the bottom boiler water is 13-17 g: 44-48 mL.
Preferably, the waste lees are byproducts in the production of white spirit, and the bottom boiler water is wastewater in the production of white spirit.
The invention also provides a method for preparing ruminant feed by using the saccharomyces boulardii or the compound bacteria, which comprises the following steps:
inoculating the saccharomyces boulardii or the compound bacteria into the fermentation medium, and performing anaerobic fermentation for 60-96 h to obtain the ruminant feed.
Preferably, the volume ratio of the saccharomyces boulardii to the boiler water in the fermentation medium is 6-9: 44-48;
the volume ratio of the composite bacteria to the boiler water in the fermentation medium is 6-9: 44-48.
Preferably, the anaerobic fermentation is fermentation in an anaerobic breath bag.
Preferably, the temperature of the anaerobic fermentation is 35-45 ℃.
The invention also provides the ruminant feed prepared by the method, wherein the ruminant feed contains 21.12-21.98 wt% of crude protein, 4.9-6.3 wt% of acid soluble protein and 12.13-13.25 wt% of acid cellulose.
The invention provides a saccharomyces boulardii and a fermentation medium thereof, a ruminant feed and a preparation method thereof, and the scheme of the invention has the following advantages:
(1) the compound bacteria have a synergistic effect, and are inoculated in the fermentation medium to obtain the feed suitable for the growth of the ruminant;
(2) according to the scheme, the waste water bottom boiler water in the white spirit production is utilized, the blank of the prior art for utilizing the bottom boiler water is filled by utilizing the components, the waste water treatment cost can be reduced by 50%, and the bottom boiler water is changed into valuable;
(3) the spent grains have large water content, cannot be stored for a long time, are low in price and cannot be consumed by an enterprise, and the method provides a new way for recycling the spent grains of the enterprise;
(4) the method of the invention adopts the anaerobic respiration bag for fermentation culture, so that the fermentation product can be completely fermented into the feed without any waste.
Deposit description
The yeast Saccharomyces boulardii is deposited in China general microbiological culture Collection center, has the address of No. 3 Xilu-1 of Beijing, Chaoyang, and the date of deposit is 2022 years, 4 months and 21 days, and the number of deposit is CGMCC No. 24750.
Detailed Description
The invention provides a Saccharomyces boulardii, which is preserved in China general microbiological culture Collection center with the Latin name of Saccharomyces boulardii, the address of No. 3 Xilu-Chen-Yang No. 1 Hospital, No. 3, the preservation date of No. 2022, 4 and 21 days, and the preservation number of CGMCC No. 24750.
The invention also provides a composite bacterium containing the saccharomyces boulardii, which also comprises lactobacillus paracasei and lactobacillus buchneri;
the initial viable bacteria concentration of the saccharomyces boulardii in the composite bacteria is 1.0-2.0 multiplied by 10 8 The cfu/mL is preferably 1.5X 10 8 cfu/mL; the initial viable bacteria concentration of the lactobacillus paracasei in the composite bacteria is 0.5-1.0 multiplied by 10 8 cfu/mL, preferably 0.75X 10 8 cfu/mL; the initial viable bacteria concentration of the lactobacillus buchneri in the composite bacteria is 1-3 multiplied by 10 7 cfu/mL, preferably 2X 10 7 cfu/mL。
In the invention, the preparation method of the saccharomyces boulardii comprises the following steps: and inoculating the strain of the saccharomyces boulardii into a saccharomyces boulardii liquid culture medium, and activating for 10-24 hours to obtain the saccharomyces boulardii. The liquid culture medium of the saccharomyces boulardii takes water as a solvent and comprises the following components in concentration: 18-22 g/L of glucose, preferably 20 g/L; peptone is 18-22 g/L, preferably 20 g/L; 8-12 g/L of yeast extract, preferably 10 g/L. The activation temperature is 28-32 ℃, and preferably 30 ℃; the rotation speed of the activation is 140-160 rpm, preferably 150 rpm; the activation time is preferably 15-19 h, and more preferably 17 h.
In the invention, the preparation method of the lactobacillus paracasei comprises the following steps: and (3) inoculating the lactobacillus paracasei strain into a lactobacillus paracasei liquid culture medium, and activating for 12-20 hours to obtain the lactobacillus paracasei strain. The lactobacillus paracasei liquid culture medium takes water as a solvent and comprises the following components in concentration: peptone is 8-12 g/L, preferably 10 g/L; 5-7 g/L of beef extract, preferably 6 g/L; 4-6 g/L of yeast extract powder, preferably 5 g/L; 7-9 g/L of glucose, preferably 8 g/L; 4-6 g/L of sodium acetate, preferably 5 g/L; 1.5-2.5 g/L ammonium citrate, preferably 2g/L ammonium citrate; 1.5-2.5 g/L potassium dihydrogen phosphate, preferably 2g/L potassium dihydrogen phosphate; tween-800.8-1.2 mL/L, preferably 1 mL/L; magnesium sulfate is 0.1-0.3 g/L, preferably 0.2 g/L; 0.04-0.06 g/L of manganese sulfate, preferably 0.05 g/L. The activation temperature is 36-38 ℃, and preferably 37 ℃; the activation time is preferably 16 h.
In the invention, the preparation method of the lactobacillus buchneri comprises the following steps: and inoculating lactobacillus buchneri into a lactobacillus buchneri liquid culture medium, and activating for 16-24 h to obtain the lactobacillus buchneri liquid culture medium. The lactobacillus buchneri liquid culture medium takes water as a solvent and comprises the following components in concentration: peptone is 8-12 g/L, preferably 10 g/L; 5-7 g/L of beef extract, preferably 6 g/L; 4-6 g/L of yeast extract powder, preferably 5 g/L; 7-9 g/L of glucose, preferably 8 g/L; 4-6 g/L of sodium acetate, preferably 5 g/L; 1.5-2.5 g/L ammonium citrate, preferably 2g/L ammonium citrate; 1.5-2.5 g/L potassium dihydrogen phosphate, preferably 2g/L potassium dihydrogen phosphate; tween-800.8-1.2 mL/L, preferably 1 mL/L; magnesium sulfate is 0.1-0.3 g/L, preferably 0.2 g/L; 0.04-0.06 g/L of manganese sulfate, preferably 0.05 g/L. The activation temperature is 36-38 ℃, and preferably 37 ℃; the activation time is preferably 20 h.
The invention also provides a fermentation medium of the saccharomyces boulardii or the compound bacteria, and the fermentation medium comprises the following components: waste lees, cottonseed meal, soybean meal, bran and pot water.
In the invention, the mass-volume ratio of the waste lees to the bottom boiler water is 35-39 g: 44-48 mL, preferably 37 g: 46 mL; the mass volume ratio of the cottonseed meal to the bottom boiler water is 24-28 g: 44-48 mL, preferably 26 g: 46 mL; the mass volume ratio of the soybean meal to the bottom boiler water is 20-24 g: 44-48 mL, preferably 22 g: 46 mL; the mass volume ratio of the bran to the bottom boiler water is 13-17 g: 44-48 mL, preferably 15 g: 46 mL.
In the invention, the waste lees are byproducts in the production of white spirit, and the bottom boiler water is waste water in the production of white spirit.
The invention also provides a method for preparing ruminant feed by using the saccharomyces boulardii or the compound bacteria, which comprises the following steps: inoculating the saccharomyces boulardii or the compound bacteria into the fermentation medium, and performing anaerobic fermentation for 60-96 h to obtain the ruminant feed.
In the invention, the volume ratio of the saccharomyces boulardii to the pot water in the fermentation medium is 6-9: 44-48, preferably 7.5: 46; the volume ratio of the complex bacteria to the boiler water in the fermentation medium is 6-9: 44-48, and preferably 7.5: 46. In the invention, the anaerobic fermentation is fermentation in an anaerobic respiration bag. In the invention, the temperature of anaerobic fermentation is 35-45 ℃, and preferably 40 ℃. In the invention, the time for anaerobic fermentation is preferably 70-86 h, and more preferably 78 h.
The invention also provides the ruminant feed prepared by the method, wherein the ruminant feed contains 21.12-21.98 wt% of crude protein, 4.9-6.3 wt% of acid soluble protein and 12.13-13.25 wt% of acid cellulose.
The embodiments of the present invention will be described in detail with reference to the following examples, but they should not be construed as limiting the scope of the present invention.
The lactobacillus paracasei strain is a strain purchased from China center for culture Collection of microorganisms (CGMCC) and has the strain number of 1.12731.
The lactobacillus buchneri strain is a strain purchased from China center for culture Collection of microorganisms (CGMCC) and the number of the strain is 1.15607.
The waste lees and the bottom boiler water in the embodiment of the invention are from strong aromatic type liquor factories in Yibin city.
All media mentioned in the examples of the present invention were used after autoclaving at 121 ℃ for 30min after the preparation.
Example 1
The preparation method of the liquid culture medium of the saccharomyces boulardii comprises the following steps: glucose 18g, peptone 20g, and yeast extract 12g were added to 1L of water to obtain.
The preparation method of the liquid culture medium of lactobacillus paracasei and lactobacillus buchneri comprises the following steps: the feed additive is prepared by dissolving 12g of peptone, 5g of beef extract, 6g of yeast extract powder, 8g of glucose, 4g of sodium acetate, 2.5g of ammonium citrate, 2g of monopotassium phosphate, 801.2mL of tween-801.2, 0.2g of magnesium sulfate and 0.04g of manganese sulfate in water and fixing the volume to 1L.
Inoculating Saccharomyces boulardii strain in Saccharomyces boulardii liquid culture medium, activating at 28 deg.C and 160rpm for 24 hr to obtain viable count of 2.0 × 10 8 cfu/mL of Saccharomyces boulardii.
Inoculating Lactobacillus paracasei strain in Lactobacillus paracasei liquid culture medium, activating at 38 deg.C for 12 hr to obtain viable count of 0.5 × 10 8 cfu/mL Lactobacillus paracasei.
Inoculating Lactobacillus buchneri in Lactobacillus buchneri liquid culture medium, activating at 36 deg.C for 16 hr to obtain viable count of 3.0 × 10 7 cfu/mL Lactobacillus buchneri.
And uniformly mixing 37g of waste lees, 24g of cottonseed meal, 24g of soybean meal and 15g of bran, and adding 46mL of bottom boiler water to obtain a fermentation culture medium for later use.
Inoculating 2mL of saccharomyces boulardii, 3mL of lactobacillus paracasei and 1mL of lactobacillus buchneri into a fermentation culture medium, uniformly mixing with the fermentation culture medium, filling into an anaerobic respiration bag, and performing anaerobic fermentation for 80h at 35 ℃ to obtain the ruminant feed.
The results of measuring the contents of protein and acidic cellulose contained in the feed for ruminants are shown in table 1.
TABLE 1 ruminant feed index detection
| Detecting the index | Crude protein | Acid soluble protein | Acid cellulose |
| (w/w) test result | 21.98 | 4.9 | 13.25 |
Table 1 shows that the crude protein content of the ruminant feed prepared by the invention is up to 21.98 wt%, which indicates that the high-protein feed can be obtained by fermentation by using the method of the invention.
Example 2
The preparation method of the liquid culture medium of the saccharomyces boulardii comprises the following steps: glucose 20g, peptone 18g, and yeast extract 10g were added to 1L of water to obtain.
The preparation method of the liquid culture medium of lactobacillus paracasei and lactobacillus buchneri comprises the following steps: 8g of peptone, 7g of beef extract, 5g of yeast extract powder, 9g of glucose, 5g of sodium acetate, 1.5g of ammonium citrate, 2.5g of monopotassium phosphate, 801mL of tween-801, 0.3g of magnesium sulfate and 0.05g of manganese sulfate, dissolving in water, and fixing the volume to 1L.
Inoculating Saccharomyces boulardii strain in Saccharomyces boulardii liquid culture medium, activating at 32 deg.C and 150rpm for 10 hr to obtain viable count of 1.0 × 10 8 cfu/mL of Saccharomyces boulardii.
Inoculating Lactobacillus paracasei strain in Lactobacillus paracasei liquid culture medium, activating at 36 deg.C for 18 hr to obtain viable count of 1.0 × 10 8 cfu/mL Lactobacillus paracasei.
Inoculating Lactobacillus buchneri into Lactobacillus buchneri liquid culture medium, activating at 38 deg.C for 18 hr to obtain viable count of 1.0 × 10 7 cfu/mL Lactobacillus buchneri.
And uniformly mixing 39g of waste lees, 25g of cottonseed meal, 22g of soybean meal and 17g of bran, and adding 48mL of bottom boiler water to obtain a fermentation culture medium for later use.
Inoculating 3mL of saccharomyces boulardii, 2mL of lactobacillus paracasei and 2mL of lactobacillus buchneri into a fermentation culture medium, uniformly mixing with the fermentation culture medium, filling into an anaerobic respiration bag, and performing anaerobic fermentation for 60 hours at 45 ℃ to obtain the ruminant feed.
The results of measuring the contents of protein and acidic cellulose contained in the feed for ruminants are shown in table 1.
TABLE 2 ruminant feed index detection
| Detecting the index | Crude protein | Acid soluble protein | Acid cellulose |
| (w/w) test result | 21.25 | 5.56 | 12.61 |
Table 2 shows that the crude protein content of the ruminant feed prepared by the method of example 2 is as high as 21.25 wt%, indicating that the method of the present invention can prepare a high protein feed.
Example 3
The preparation method of the liquid culture medium of the saccharomyces boulardii comprises the following steps: glucose 22g, peptone 22g, and yeast extract 8g were added to 1L of water to obtain.
The preparation method of the liquid culture medium of lactobacillus paracasei and lactobacillus buchneri comprises the following steps: 10g of peptone, 6g of beef extract, 4g of yeast extract powder, 7g of glucose, 6g of sodium acetate, 2g of ammonium citrate, 1.5g of monopotassium phosphate, 800.8mL of tween-800, 0.1g of magnesium sulfate and 0.06g of manganese sulfate, dissolving in water, and fixing the volume to 1L.
Inoculating Saccharomyces boulardii strain in Saccharomyces boulardii liquid culture medium, activating at 30 deg.C under 140rpm for 24 hr to obtain viable count of 1.5 × 10 8 cfu/mL of Saccharomyces boulardii.
Inoculating Lactobacillus paracasei strain in Lactobacillus paracasei liquid culture medium, activating at 37 deg.C for 20 hr to obtain viable count of 1.0 × 10 8 cfu/mL) Lactobacillus paracasei.
Inoculating Lactobacillus buchneri in Lactobacillus buchneri liquid culture medium, activating at 37 deg.C for 24 hr to obtain viable count of 2.0 × 10 7 cfu/mL Lactobacillus buchneri.
Uniformly mixing 35g of waste lees, 28g of cottonseed meal, 20g of soybean meal and 13g of bran, and adding 44mL of bottom boiler water to obtain a fermentation culture medium for later use.
Inoculating 3mL of saccharomyces boulardii, 3mL of lactobacillus paracasei and 3mL of lactobacillus buchneri into a fermentation culture medium, uniformly mixing with the fermentation culture medium, filling into an anaerobic respiration bag, and performing anaerobic fermentation for 96 hours at 40 ℃ to obtain the ruminant feed.
The results of measuring the contents of protein and acidic cellulose contained in the feed for ruminants are shown in table 1.
TABLE 3 ruminant feed index detection
| Detecting the index | Crude protein | Acid soluble protein | Acid cellulose |
| (w/w) test result | 21.12 | 6.30 | 12.13 |
Table 3 shows that the crude protein content of the ruminant feed prepared by the method of example 3 is as high as 21.12 wt%, indicating that the method of the present invention can prepare a high-protein ruminant feed.
According to the embodiments, the invention provides the saccharomyces boulardii and the fermentation medium thereof, the ruminant feed and the preparation method thereof, the scheme of the invention fills the shortage of the prior art in the utilization of the pot water in the white spirit production process, reduces the cost for treating the pot water, and can change the pot water into valuable. The scheme of the invention also recycles the waste lees generated in the production process of the white spirit, and provides a new way for the utilization of the waste lees. The method of the invention also utilizes the anaerobic respiration bag to ferment the feed, so that products in the fermentation process are all fermented into the feed without any waste.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
Claims (10)
1. The Saccharomyces boulardii is deposited in China general microbiological culture Collection center with the address of No. 3 Xilu-Beijing-oriented Xingyang No. 1 Beijing, the preservation date of No. 21-4 months in 2022 and the preservation number of (CGMCC No. 24750).
2. A composite bacterium comprising the saccharomyces boulardii of claim 1, further comprising lactobacillus paracasei and lactobacillus buchneri;
the initial viable bacteria concentration of the saccharomyces boulardii in the composite bacteria is 1.0-2.0 multiplied by 10 8 cfu/mL；
The initial viable bacteria concentration of the lactobacillus paracasei in the composite bacteria is 0.5-1.0 multiplied by 10 8 cfu/mL；
The initial viable bacteria concentration of the lactobacillus buchneri in the composite bacteria is 1-3 multiplied by 10 7 cfu/mL。
3. A fermentation medium of the saccharomyces boulardii of claim 1 or the complex bacteria of claim 2, which comprises the following components:
waste lees, cottonseed meal, soybean meal, bran and pot bottom water.
4. The fermentation medium of claim 3, wherein the mass-to-volume ratio of the spent grains to the water in the bottom boiler is 35-39 g: 44-48 mL;
the mass volume ratio of the cottonseed meal to the bottom boiler water is 24-28 g: 44-48 mL;
the mass volume ratio of the soybean meal to the bottom boiler water is 20-24 g: 44-48 mL;
the mass volume ratio of the bran to the bottom boiler water is 13-17 g: 44-48 mL.
5. The fermentation medium of claim 3 or 4, wherein the spent grain is a byproduct in the production of white spirit, and the bottom boiler water is waste water in the production of white spirit.
6. A method for preparing a ruminant feed by using the Saccharomyces boulardii of claim 1 or the composite bacterium of claim 2, comprising the steps of:
inoculating the Saccharomyces boulardii or the composite bacterium of claim 1 or 2 into the fermentation medium of any one of claims 3 to 5, and performing anaerobic fermentation for 60 to 96 hours to obtain the ruminant feed.
7. The method according to claim 6, wherein the volume ratio of the Saccharomyces boulardii to the boiler water in the fermentation medium is 6-9: 44-48;
the volume ratio of the composite bacteria to the boiler water in the fermentation medium is 6-9: 44-48.
8. The method of claim 7, wherein the anaerobic fermentation is fermentation in an anaerobic respiration bag.
9. The method according to claim 8, wherein the temperature of the anaerobic fermentation is 35-45 ℃.
10. The ruminant feed prepared by the method of any one of claims 6 to 9, wherein the ruminant feed contains 21.12 to 21.98 wt% of crude protein, 4.9 to 6.3 wt% of acid soluble protein and 12.13 to 13.25 wt% of acid cellulose.
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