Method for reducing bitter taste of waste beer yeast liquid and application

Abstract

The invention provides a method for reducing bitter taste of waste beer yeast liquid and application, and belongs to the technical field of microorganisms, a biological fermentation technology is adopted, a specific microorganism-bacillus subtilis is used for fermentation, bitter substances are degraded, specific metabolites are metabolized, and the bitter taste of the waste beer yeast liquid is reduced. Protein (about 40-60% of dry basis), B vitamins (especially B1, B2 and nicotinic acid), minerals (selenium, chromium and zinc), nucleic acid (RNA and DNA), beta-glucan and the like in the waste beer yeast liquid are reserved, and the waste beer yeast liquid can be used as a functional animal feed additive.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, and in particular relates to a method and application for reducing the bitterness of waste brewer's yeast liquid. Background Technology

[0002] After beer fermentation and aging, the yeast sludge that settles at the bottom of the tank undergoes repeated recycling. However, the yeast activity decreases, the mutation rate increases, and it becomes susceptible to contamination. Alternatively, due to process requirements, this portion of yeast may no longer be suitable for fermentation and becomes waste beer yeast. Statistics show that approximately 1 kilogram of waste is generated for every 5 liters of beer produced. With a global annual beer production of 19.3 million kiloliters, this corresponds to a yeast waste volume of up to 39 million tons per year.

[0003] Settled waste yeast sludge is rich in protein (about 40-60% dry basis), B vitamins (especially B1, B2, and niacin), minerals (selenium, chromium, and zinc), nucleic acids (RNA and DNA), and β-glucan, making it an ideal source of feed protein. However, its poor palatability, due to residual bitter substances (bitter taste caused by hop resin and fermentation byproducts) and coarse texture (hard cell walls of untreated yeast), limits its direct application.

[0004] The bitterness of waste yeast sludge mainly consists of the following three parts: ① isomers of α-acids (iso-α-acids) and their derivatives from hop resins. ② oxides of β-acids and their derivatives from hop resins. ③ Some bitter metal salts, primarily magnesium salts. Currently, there is an urgent need for a method to eliminate bitter substances, reduce the content of bitter substances in yeast sludge, and improve the utilization rate of yeast sludge. Summary of the Invention

[0005] In view of this, the purpose of the present invention is to provide a method and application for reducing the bitterness of beer waste yeast liquid.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution:

[0007] This invention provides a method for reducing the bitterness of waste brewer's yeast liquid, which involves inoculating Bacillus subtilis into a fermentation medium and fermenting it.

[0008] The fermentation medium comprises beer waste yeast liquid with a volume concentration of 10% to 45%.

[0009] Preferably, the inoculum size of Bacillus subtilis is 0.5–2% of the fermentation medium volume, and the viable count of Bacillus subtilis is 2 × 10⁻⁶. 9 ~3×10 9 CFU / mL.

[0010] Preferably, the Bacillus subtilis is Bacillus subtilis YB18, with accession number CGMCC No. 17642.

[0011] Preferably, the Bacillus subtilis is activated before inoculation. The activation method is to inoculate Bacillus subtilis into a liquid culture medium and culture it at 35-40°C and 100-200 rpm for 12-24 hours.

[0012] The liquid culture medium comprises the following components at the following concentrations: peptone 5–15 g / L, beef extract 1–5 g / L, glucose 15–25 g / L, and sodium chloride 3–7 g / L.

[0013] Preferably, the fermentation medium further includes components at the following concentrations:

[0014] Monosodium glutamate 30-50 g / L, peptone 3-7 g / L, sucrose 30-50 g / L, sodium chloride 5-15 g / L.

[0015] Preferably, the fermentation temperature is 35–40°C.

[0016] Preferably, the fermentation speed is 100-200 rpm.

[0017] Preferably, the fermentation time is 8 to 96 hours.

[0018] This invention provides a low-bitterness beer waste yeast liquid prepared by the method described above.

[0019] This invention provides the application of the low-bitterness beer waste yeast liquid in the preparation of feed additives.

[0020] Compared with the prior art, the present invention has the following beneficial effects:

[0021] This invention provides a method for reducing the bitterness of brewer's waste yeast liquid. It employs bio-fermentation technology and utilizes a specific microorganism, Bacillus subtilis, for fermentation. While degrading bitter substances, it also metabolizes specific metabolites and retains proteins (approximately 40-60% dry basis), B vitamins (especially B1, B2, and niacin), minerals (selenium, chromium, and zinc), nucleic acids (RNA and DNA), β-glucan, etc., in the brewer's waste yeast liquid. It can be used as a functional animal feed additive. Attached Figure Description

[0022] Figure 1 Fermentation conditions under different contents of waste beer yeast in the fermentation substrate;

[0023] Figure 2 The bar chart shows the effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in the fermentation medium containing 10% beer waste yeast liquid.

[0024] Figure 3The bar chart shows the effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in the fermentation medium containing 20% ​​beer waste yeast liquid.

[0025] Figure 4 Bar chart showing the effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in fermentation medium containing 30% beer waste yeast liquid;

[0026] Figure 5 The bar chart shows the effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in the fermentation medium containing 40% beer waste yeast liquid.

[0027] Biological Preservation Instructions

[0028] The Bacillus subtilis YB18 is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 17642, classified as Bacillus subtilis, and deposited on April 28, 2019. Detailed Implementation

[0029] This invention provides a method for reducing the bitterness of waste brewer's yeast liquid, which involves inoculating Bacillus subtilis into a fermentation medium and fermenting it.

[0030] The fermentation medium includes waste brewer's yeast liquid.

[0031] In this invention, the Bacillus subtilis is activated before inoculation. The preferred activation method is to inoculate Bacillus subtilis into a liquid culture medium for cultivation. The preferred cultivation temperature is 35-40°C, more preferably 36-39°C, and even more preferably 37°C. The preferred cultivation speed is 100-200 rpm, more preferably 120-180 rpm, and even more preferably 150 rpm. The preferred cultivation time is 12-24 h, more preferably 14-22 h, and even more preferably 18 h.

[0032] In this invention, the liquid culture medium comprises components at the following concentrations:

[0033] The peptone concentration is preferably 5-15 g / L, more preferably 7-12 g / L, and even more preferably 15 g / L;

[0034] The beef extract powder is preferably 1-5 g / L, more preferably 2-4 g / L, and even more preferably 3 g / L;

[0035] The glucose concentration is preferably 15-25 g / L, more preferably 17-22 g / L, and even more preferably 20 g / L;

[0036] The sodium chloride concentration is preferably 3-7 g / L, more preferably 4-6 g / L, and even more preferably 5 g / L.

[0037] In this invention, the Bacillus subtilis is Bacillus subtilis YB18, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 17642, classified as Bacillus subtilis, deposited on April 28, 2019, and published in CN202411928397.0.

[0038] In this invention, the inoculum amount of Bacillus subtilis is preferably 0.5% to 2% of the fermentation medium volume, more preferably 0.7% to 1.5%, and even more preferably 1%. The viable count of Bacillus subtilis is preferably 2 × 10⁻⁶. 9 ~3×10 9 CFU / mL, further preferably 2.2 × 10⁻⁶. 9 CFU / mL ~ 2.8 × 10 9 CFU / mL, more preferably 2.6 × 10⁻⁶. 9 CFU / mL.

[0039] In this invention, the fermentation medium comprises waste brewer's yeast liquid, wherein the volume concentration of the waste brewer's yeast liquid is 10% to 45%, preferably 20% to 42%, and more preferably 40%. The fermentation medium also includes components at the following concentrations:

[0040] The monosodium glutamate (MSG) concentration is preferably 30–50 g / L, more preferably 35–45 g / L, and even more preferably 40 g / L.

[0041] The peptone concentration is preferably 3-7 g / L, more preferably 4-6 g / L, and even more preferably 5 g / L;

[0042] The sucrose concentration is preferably 30–50 g / L, more preferably 35–45 g / L, and even more preferably 40 g / L;

[0043] The sodium chloride concentration is preferably 5–15 g / L, more preferably 7–12 g / L, and even more preferably 10 g / L.

[0044] In this invention, Bacillus subtilis is inoculated into a fermentation medium containing waste brewer's yeast liquid, and fermentation is carried out. The fermentation temperature is preferably 35-40°C, more preferably 36-39°C, and even more preferably 37°C. The fermentation speed is preferably 100-200 rpm, more preferably 120-180 rpm, and even more preferably 150 rpm. The fermentation time is preferably 8-96 hours, more preferably 24-72 hours, and even more preferably 48 hours.

[0045] This invention provides a low-bitterness beer waste yeast liquid prepared by the method described above.

[0046] This invention provides the application of the low-bitterness beer waste yeast liquid in the preparation of feed additives.

[0047] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0048] Methods for detecting bitter substances:

[0049] Add 10 mL of sample to a 100 mL centrifuge tube, along with 1 drop of n-octanol (antifoaming agent), 0.5 mL of 6 mol / L hydrochloric acid, and 20 mL of isooctane. Cap the tube and adjust the shaker frequency to 775 rpm for 3 minutes. Centrifuge at 5000 rpm for 10 minutes. After centrifugation, collect the supernatant and use a microplate reader. Using isooctane as a blank, measure the absorbance at 279 nm using a quartz cuvette to determine the relative content.

[0050] The formula for calculating the content of bitter substances in beer is shown in equation (1):

[0051] X = A279 × 50

[0052] In formula (1):

[0053] The content of bitter substances in sample X is expressed in "BU" units;

[0054] A279 - The absorbance of the sample was measured at a wavelength of 279 nm;

[0055] 50 - Conversion factor.

[0056] Example 1

[0057] One loopful of Bacillus subtilis YB18 colonies preserved on a slant was picked and inoculated into a 250 mL Erlenmeyer flask containing 50 mL of liquid culture medium (unit: peptone 10, beef extract 3, glucose 20, sodium chloride 5 g / L). The flask was incubated at 37℃ and 150 rpm for 18 h to obtain a mature Bacillus subtilis seed culture with a viable count of 2.6 × 10⁻⁶. 9CFU / mL.

[0058] Example 2

[0059] At an inoculation rate of 2%, the Bacillus subtilis seed culture prepared in Example 1 was inoculated into fermentation medium containing 10%, 20%, 30%, and 40% volume of waste brewer's yeast liquid (MSG 40 g / L, peptone 5 g / L, sucrose 40 g / L, sodium chloride 10 g / L, liquid volume 50 mL / 250 mL). Fermentation was carried out at 37°C and 150 rpm for 48 h. The results are detailed in [link to results]. Figure 1 The results showed:

[0060] The fermentation medium containing 10% brewer's waste yeast liquid had a γ-polyglutamic acid content of 10.66 g / L and a bitter substance content of 10.43 BU (a decrease of 6.57% compared to before fermentation).

[0061] The fermentation broth containing 20% ​​waste brewer's yeast contained 9.21 g / L of γ-polyglutamic acid and 15.67 BU of bitter substances (a decrease of 37.58% compared to before fermentation).

[0062] The fermentation broth containing 30% waste brewer's yeast contained 6.66 g / L of γ-polyglutamic acid and 16.50 BU of bitter substances (a decrease of 55.04% compared to before fermentation).

[0063] The fermentation broth containing 40% waste brewer's yeast contained 3.12 g / L of γ-polyglutamic acid and 24.07 BU of bitter substances (a decrease of 54.01% compared to before fermentation).

[0064] Example 3

[0065] Effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in fermentation medium containing 10% brewer's yeast broth

[0066] At an inoculation rate of 2%, the Bacillus subtilis seed culture prepared in Example 1 was inoculated into a fermentation medium containing 10% brewer's yeast liquid (MSG 40 g / L, peptone 5 g / L, sucrose 40 g / L, sodium chloride 10 g / L, liquid volume 50 mL / 250 mL). Fermentation was carried out at 37 °C and 150 rpm. The contents of γ-polyglutamic acid and bitter substances were detected at fermentation times of 24 h, 48 h and 72 h, respectively.

[0067] The results are as follows Figure 2 As shown: After 24 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 4.72 g / L, and the content of bitter substances was 5.65 BU (a decrease of 49.22% compared with before fermentation).

[0068] After 48 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 10.66 g / L, and the content of bitter substances was 10.43 BU (a decrease of 5.76% compared with before fermentation).

[0069] After 72 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 13.67 g / L, and the content of bitter substances was 10.53 BU (a decrease of 4.85% compared with before fermentation).

[0070] When the fermentation time is 24 hours, the fermentation medium containing 10% waste brewer's yeast liquid has the best effect on reducing bitterness.

[0071] Example 4

[0072] Effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in fermentation medium containing 20% ​​brewer's yeast broth

[0073] At an inoculation rate of 2%, the Bacillus subtilis seed culture prepared in Example 1 was inoculated into a fermentation medium containing 20% ​​brewer's yeast liquid (MSG 40 g / L, peptone 5 g / L, sucrose 40 g / L, sodium chloride 10 g / L, liquid volume 50 mL / 250 mL). Fermentation was carried out at 37 °C and 150 rpm. The contents of γ-polyglutamic acid and bitter substances were detected at fermentation times of 24 h, 48 h and 72 h, respectively.

[0074] like Figure 3 As shown, after 24 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 2.45 g / L, and the content of bitter substances was 13.10 BU (a decrease of 47.81% compared with before fermentation).

[0075] After 48 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 9.21 g / L, and the content of bitter substances was 15.67 BU (a decrease of 37.58% compared with before fermentation).

[0076] After 72 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 10.02 g / L, and the content of bitter substances was 15.71 BU (a decrease of 37.41% compared with before fermentation).

[0077] When the fermentation time is 24 hours, the fermentation medium containing 20% ​​waste brewer's yeast liquid has the best effect on reducing bitterness.

[0078] Example 5

[0079] Effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in fermentation medium containing 30% brewer's waste yeast liquid

[0080] At an inoculation rate of 2%, the Bacillus subtilis seed culture prepared in Example 1 was inoculated into a fermentation medium containing 30% brewer's yeast liquid (MSG 40 g / L, peptone 5 g / L, sucrose 40 g / L, sodium chloride 10 g / L, liquid volume 50 mL / 250 mL). Fermentation was carried out at 37 °C and 150 rpm. The contents of γ-polyglutamic acid and bitter substances were detected at fermentation times of 24 h, 48 h and 72 h, respectively.

[0081] The results are as follows Figure 4 As shown, after 24 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 2.1 g / L, and the content of bitter substances was 28.21 BU (a decrease of 23.13% compared with before fermentation).

[0082] After 48 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 6.66 g / L, and the content of bitter substances was 16.50 BU (a decrease of 55.04% compared with before fermentation).

[0083] After 72 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 8.34 g / L, and the content of bitter substances was 16.51 BU (a decrease of 55.01% compared with before fermentation).

[0084] When the fermentation time is 48 hours, the fermentation medium containing 30% waste brewer's yeast liquid has the best effect on reducing bitterness.

[0085] Example 6

[0086] Effect of fermentation time on the content of γ-polyglutamic acid and bitter substances in fermentation medium containing 40% brewer's yeast broth

[0087] At an inoculation rate of 2%, the Bacillus subtilis seed culture prepared in Example 1 was inoculated into a fermentation medium containing 40% brewer's yeast liquid (MSG 40 g / L, peptone 5 g / L, sucrose 40 g / L, sodium chloride 10 g / L, liquid volume 50 mL / 250 mL). Fermentation was carried out at 37 °C and 150 rpm. The contents of γ-polyglutamic acid and bitter substances were detected at fermentation times of 24 h, 48 h and 72 h, respectively.

[0088] The results are as follows Figure 5 As shown: After 24 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 1.45 g / L, and the content of bitter substances was 48.33 BU (a decrease of 7.64% compared with before fermentation).

[0089] After 48 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 3.12 g / L, and the content of bitter substances was 24.07 BU (a decrease of 54.01% compared with before fermentation).

[0090] After 72 hours of fermentation, the content of γ-polyglutamic acid in the fermentation medium was 4.17 g / L, and the content of bitter substances was 25.07 BU (a decrease of 52.10% compared with before fermentation).

[0091] When the fermentation time is 48 hours, the fermentation medium containing 40% waste brewer's yeast liquid has the best effect on reducing bitterness.

[0092] Figures 1-5 The study indicated that as fermentation time increased, the content of γ-polyglutamic acid in the fermentation broth gradually increased, while the content of bitter substances showed a trend of first decreasing and then increasing. This suggests that the elimination of bitter substances is somewhat correlated with the concentration of γ-polyglutamic acid. Both higher and lower concentrations of γ-polyglutamic acid are detrimental to the elimination of bitter substances in beer waste yeast broth. This may be because during the microbial fermentation and synthesis of γ-polyglutamic acid, the content of γ-polyglutamic acid in the fermentation broth increases from low to high, and its molecular weight increases from low to high. A suitable amount of low molecular weight γ-polyglutamic acid is more conducive to binding with bitter substances, resulting in a reduction of bitter substances in the fermentation broth. However, when a large amount of high molecular weight γ-polyglutamic acid appears, hydrogen bonds are formed between γ-polyglutamic acid molecules, preventing them from binding with bitter substances, resulting in an insignificant reduction in bitter substances in the fermentation broth. Among these, the content of bitter substances was lowest when the fermentation time was 48 hours.

[0093] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for reducing the bitterness of spent brewer's yeast liquid, characterized in that, Bacillus subtilis was inoculated into a fermentation medium and fermented. The fermentation medium comprises beer waste yeast liquid with a volume concentration of 10% to 45%.

2. The method according to claim 1, characterized in that, The inoculum size of Bacillus subtilis is 0.5–2% of the fermentation medium volume, and the viable count of Bacillus subtilis is 2 × 10⁻⁶. 9 ~3×10 9 CFU / mL.

3. The method according to claim 1, characterized in that, The Bacillus subtilis strain mentioned is Bacillus subtilis YB18, with accession number CGMCCNo.17642.

4. The method according to claim 3, characterized in that, The Bacillus subtilis was activated before inoculation. The activation method was to inoculate Bacillus subtilis into a liquid culture medium and incubate it at 35-40°C and 100-200 rpm for 12-24 hours. The liquid culture medium comprises the following components at the following concentrations: peptone 5–15 g / L, beef extract 1–5 g / L, glucose 15–25 g / L, and sodium chloride 3–7 g / L.

5. The method according to claim 1, characterized in that, The fermentation medium also includes components at the following concentrations: Monosodium glutamate 30-50 g / L, peptone 3-7 g / L, sucrose 30-50 g / L, sodium chloride 5-15 g / L.

6. The method according to claim 1, characterized in that, The fermentation temperature is 35–40°C.

7. The method according to claim 6, characterized in that, The fermentation speed is 100-200 rpm.

8. The method according to claim 7, characterized in that, The fermentation time is 8 to 96 hours.

9. The low-bitterness beer waste yeast liquid prepared by any one of claims 1 to 8.

10. The application of the low-bitterness beer waste yeast liquid according to claim 9 in the preparation of feed additives.

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