A method for producing lactic acid by fermenting barley malt residue

Abstract

The present invention relates to a method for producing lactic acid by fermenting barley malt residue, which specifically comprises the following steps: (1) adding water to the barley malt residue after alkali pretreatment to adjust the solid content of the fermentation production system to 10%-15%, adding cellulase at an addition amount of 5-10 FPU / g barley malt residue, and hydrolyzing for 20-48 hours at 50°C-60°C and pH 4.5-5.2 to obtain a prehydrolyzed substrate; (2) inoculating the hydrolyzate obtained in step (1) with a live bacterial count of 1×10 8 ‑1×10 10 CFU / mL of lactic acid bacteria seed liquid is fermented at 40-55°C and pH 6.0-7.2 for 72-96 hours to obtain a fermentation liquid. The commercial cellulase dosage of the invention is low, the lactic acid production rate and conversion rate are high, and a new way is provided for the cheap industrial production of lactic acid.

Description

Technical Field

[0001] The invention relates to the technical field of biomass waste resource utilization, and in particular to a method for producing lactic acid by fermenting barley malt residue. Background Art

[0002] Barley malt residue (BSG) is a waste biomass raw material generated by the use of barley in industrial production processes, including residues from industrial brewing, beverage processing, food production and other processes and their processing residues. Currently, about 20 kg of BSG is produced as a by-product for every 100 L of beer produced. It is estimated that more than 30 million tons of waste BSG are produced worldwide each year, and this number is expected to increase further with the booming microbrewery market. Currently, most BSG is directly discarded to landfills or used as animal feed. BSG is rich in insoluble fibers, namely the husk, seed coat and pericarp, and storage proteins of endosperm cells, which account for about 70% and 20% of its composition, respectively. The fibers are mainly composed of cellulose, hemicellulose (i.e., arabinoxylan) and lignin. Due to its high carbohydrate content (up to 50% dry weight), waste BSG biomass raw materials are attractive for biofermentation production processes such as energy and biofuels, biogas, antibiotics, enzyme preparations and some important organic acids. It is a highly promising lignocellulosic biomass raw material that can be used for the bioconversion production of various high-value-added platform chemicals.

[0003] In existing research, BSG is used to prepare lactic acid, but there are still technical bottlenecks: first, it is necessary to add exogenous nutrients such as nitrogen sources, metal ions and trace elements, and additionally add β-glucosidase to maintain a high substrate hydrolysis efficiency, which leads to increased costs; second, the current BSG fermentation mainly adopts a synchronous hydrolysis and saccharification mode, and the initial fermentable sugar concentration of the system is too low, resulting in slow growth and metabolism of lactic acid bacteria, which is not conducive to lactic acid bioconversion; third, the hydrolysis product of BSG is not only composed of hexose (such as glucose and cellobiose, etc.), but also releases pentose (such as xylose and arabinose, etc.) with a comparable concentration. Glucose and other hexose are preferentially used by lactic acid bacteria as the preferred carbon source and inhibit the catabolism of other sugars such as pentose, resulting in delayed lactic acid production and long fermentation time, and low production rate and yield. Therefore, it is necessary to study a more efficient and economical BSG lactic acid fermentation method. Summary of the invention

[0004] The object of the present invention is to provide a method for efficiently fermenting barley malt residue to produce lactic acid, which specifically comprises the following steps:

[0005] (1) adding water to the alkali-pretreated barley malt residue to adjust the solid content of the fermentation production system to 10%-15%, adding cellulase at an addition amount of 5-10 FPU / g barley malt residue, and hydrolyzing at 50°C-60°C and pH 4.5-5.2 for 20-48 hours to obtain a prehydrolyzed substrate;

[0006] (2) The hydrolyzate obtained in step (1) was inoculated with a live bacterial count of 1×10 8 -1×10 10 The lactic acid bacteria seed liquid with a CFU / mL is fermented at 40-55° C. and pH 6.0-7.2 for 72-96 hours to obtain a fermentation liquid.

[0007] In a preferred technical solution of the present invention, the barley malt residue is a waste biomass raw material generated by using barley in an industrial production process, including residues from industrial brewing, beverage processing, and food production processes and processing residues thereof.

[0008] In a preferred technical solution of the present invention, in the barley malt residue after alkali pretreatment, the cellulose content is 15%-30% of the barley malt residue dry basis, and the hemicellulose content is 20%-40% of the barley malt residue dry basis.

[0009] In the preferred technical scheme of the present invention, in the step (1), the preparation method of the barley malt residue after alkali pretreatment is to add alkali solution with a concentration of 0.5-3.0wt% to the barley malt residue at a solid-liquid weight ratio of 1:5-1:10, cook at 100°C-120°C for 10-20 min, separate the solid and liquid, and wash the solid residue with water until it is neutral.

[0010] In a preferred technical solution of the present invention, the alkali solution is any one of NaOH and KOH or a combination thereof.

[0011] In the preferred technical solution of the present invention, in step (2), the method for preparing the lactic acid bacteria seed liquid is: Enterococcus mundtii CGMCC 22227 glycerol frozen bacteria were transferred to MRS medium and incubated for 24-48 hours to obtain activated seed solution. Then, the activated seed solution was transferred into mMRS medium at an inoculum volume of 10-15%, and anaerobically cultured at 40℃-45℃ for 8-12 hours to obtain a viable cell count of 1×10 8 -1×10 10 CFU / mL of lactic acid bacteria seed solution.

[0012] In the preferred technical scheme of the present invention, the composition of the mMRS medium is: per liter of deionized water contains 10g peptone, 8g beer extract, 5g CH3COONa·3H2O, 4g yeast extract, 2g K2HPO4, 2g ammonium citrate, 0.2gMgSO4·7H2O, 0.05g MnSO4·4H2O, 1 mL Tween 80, 10g cellobiose and 10g xylose, the initial pH value is adjusted to 7.0, and sterilized at 115°C for 15 min.

[0013] In a preferred technical solution of the present invention, in the step (2), the fermentation temperature is 40°C-45°C.

[0014] In a preferred technical solution of the present invention, the lactic acid concentration in the fermentation broth is not less than 45 g / L, preferably not less than 50 g / L.

[0015] In a preferred technical solution of the present invention, the lactic acid conversion rate of the fermentable sugar in the substrate is not less than 0.60 g / g, preferably not less than 0.7 g / g.

[0016] In the preferred technical solution of the present invention, the lactic acid production rate is not less than 3.0 g / h / L, preferably not less than 3.5 g / h / L.

[0017] In a preferred technical solution of the present invention, the acetic acid concentration in the fermentation broth is less than 2.0 g / L, preferably less than 1.3 g / L.

[0018] In a preferred technical solution of the present invention, the lactic acid output per unit of enzyme preparation added is greater than 9 (g-Lac / FPU), preferably greater than 10 (g-Lac / FPU).

[0019] Unless otherwise specified, when the present invention relates to the percentage between liquids, the percentage is volume / volume percentage; when the present invention relates to the percentage between liquids and solids, the percentage is volume / weight percentage; when the present invention relates to the percentage between solids and liquids, the percentage is weight / volume percentage; the rest are weight / weight percentages.

[0020] Unless otherwise stated, the present invention uses the following method to detect the content of fermentation products:

[0021] (1) The concentrations of lactic acid and acetic acid were determined by high performance liquid chromatography, using a Shodex Sugar SH1011 liquid chromatography column (8.0 mm × 300 mm) to separate the samples, and then entering the RID detector for detection. The chromatographic conditions were: column temperature 60°C; mobile phase 5 mM H2SO4; flow rate 1.0 mL / min; injection volume 10 μL.

[0022] (2) Substrate fermentable sugar lactic acid conversion rate = maximum lactic acid concentration / total sugar concentration

[0023] The total sugar concentration was determined using the phenol-sulfuric acid method. First, use standard glucose to make a standard curve. The specific method is as follows: accurately weigh 10 mg of standard glucose in a 250 mL volumetric flask, add water to the scale, and absorb 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.8 and 1.0 mL respectively, then add distilled water to 1.0 mL, then add 1.0 mL of 5% phenol solution and 5.0 mL of concentrated sulfuric acid, shake well and cool, place at room temperature for 20 min, measure the optical density at 490 nm, use 1.0 mL of water as a blank according to the same color development operation, the horizontal axis is the microgram number of total sugar, and the vertical axis is the optical density value to obtain the standard curve. The sample processing is consistent with the above operation, and the total sugar content in the sample is finally obtained according to the standard curve.

[0024] (3) Lactic acid production rate = change in lactic acid concentration during fermentation / fermentation time.

[0025] (4) Lactic acid production per unit of enzyme preparation added = maximum lactic acid concentration / amount of cellulose added.

[0026] (5) The cellulose and hemicellulose contents were quantitatively determined using the National Renewable Energy Laboratory (NREL) method.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] 1. The present invention uses alkali-pretreated barley malt residue as raw material, and only adds low-content cellulase for pre-hydrolysis, so that more than 50% of the cellulose in the raw material is converted into glucose and cellobiose in the process, and then directly inoculates lactic acid bacteria for synchronous fermentation, which solves the feedback inhibition effect of the enzymatic hydrolysis process, improves the substrate lactic acid conversion rate, realizes the synchronous lactic acid bioconversion of the mixed substrate of xylose, arabinose (pentose), glucose (hexose), and cellobiose, and improves the lactic acid production rate.

[0029] 2. The present invention does not require the addition of β-glucosidase and other nutrients, the dosage of commercial cellulase is low, the lactic acid production rate and conversion rate are high, and its production cost is 20%-40% lower than the conventional pretreatment-enzymatic saccharification-fermentation lactic acid production method, providing a new way for the cheap industrial production of lactic acid.

[0030] 3. The process of the present invention is simple, low in cost, environmentally friendly, and can be industrially produced. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Comparison of lactic acid production in the fermentation broth of Examples 1-3 and Comparative Examples 1-2.

[0032] Figure 2 Comparison of lactic acid production rates during fermentation of Examples 1-3 and Comparative Examples 1-2.

[0033] Figure 3 Comparison of acetic acid concentration in the fermentation broth of Example 1-3 and Comparative Example 1-2.

[0034] Figure 4 Comparison of lactic acid production per unit of enzyme preparation added in Example 1-3 and Comparative Example 1-2.

[0035] Figure 5 Comparison of the conversion rates of fermentable sugars and lactic acid in Examples 1-3 and Comparative Examples 1-2. DETAILED DESCRIPTION

[0036] The following examples are used to illustrate the present invention, but they are not intended to limit the scope of the present invention.

[0037] Take the discarded barley malt residue (water content 60-70%) from the brewery, then add 1.0wt% NaOH alkali solution, adjust the solid-liquid weight ratio to 1:10 and mix evenly, cook at 120℃ for 15 min, after solid-liquid separation, discard the liquid, wash the solid residue with water until neutral, and obtain the barley malt residue after alkali pretreatment. According to the test, the barley malt residue after alkali pretreatment contains 18.2% cellulose and 20.4% hemicellulose on a dry basis.

[0038] Enterococcus monnieri Enterococcus mundtii CGMCC 22227. The steps of culturing lactic acid bacteria seed solution are as follows: 1 mL of Enterococcus mundtii The CGMCC 22227 glycerol-frozen bacteria were transferred to 9 mL of mMRS medium and incubated at 43°C for 24 h to obtain activated seed solution. Then, 10 mL of the activated seed solution was transferred to 90 mL of mMRS medium and anaerobically cultured at 43°C for 8 h to obtain lactic acid bacteria seed solution with a viable count of 3.7 × 10 8 CFU / mL.

[0039] mMRS medium contains the following ingredients per liter of deionized water: 10 g peptone, 8 g beer extract, 5 g CH3COONa·3H2O, 4 g yeast extract, 2 g K2HPO4, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 gMnSO4·4H2O, 1 mL Tween 80, 10 g cellobiose and 10 g xylose, the initial pH value was adjusted to 7.0, and sterilized at 115°C for 15 min.

[0040] Cellulase was commercially available and had an enzyme activity of 60 FPU / mL.

[0041] Example 1 Method for efficiently fermenting barley malt residue to produce lactic acid

[0042] (1) Take 20 g of barley malt residue after alkali pretreatment, add water to adjust the solid content of the fermentation production system to 10%, add cellulase at an addition amount of 5 FPU / g barley malt residue, and hydrolyze at 50°C and pH 5.0 for 20 hours to obtain a prehydrolyzed substrate;

[0043] (2) Adding the lactic acid bacteria seed solution at an inoculum amount of 10% (v / v) to the pre-hydrolyzed substrate of step (1), and fermenting at 43° C. and pH 6.0 for 96 hours to obtain a fermentation liquid.

[0044] Example 2 Method for efficiently fermenting barley malt residue to produce lactic acid

[0045] (1) Take 20 g of barley malt residue after alkali pretreatment, add water to adjust the solid content of the fermentation production system to 10%, add cellulase at an addition amount of 7.5 FPU / g barley malt residue, and hydrolyze at 50°C and pH 5.0 for 30 hours to obtain a prehydrolyzed substrate;

[0046] (2) Adding the lactic acid bacteria seed solution at an inoculum amount of 10% (v / v) to the pre-hydrolyzed substrate of step (1), and fermenting at 43° C. and pH 6.0 for 84 hours to obtain a fermentation liquid.

[0047] Example 3 Method for efficiently fermenting barley malt residue to produce lactic acid

[0048] (1) Take 20 g of barley malt residue after alkali pretreatment, add water to adjust the solid content of the fermentation production system to 10%, add cellulase at an addition amount of 10 FPU / g barley malt residue, and hydrolyze at 50°C and pH 5.0 for 40 hours to obtain a hydrolyzed liquid;

[0049] (2) Adding the lactic acid bacteria seed solution at an inoculum amount of 10% (v / v) to the hydrolyzate of step (1), and fermenting at 43° C. and pH 6.0 for 72 hours to obtain a fermentation solution.

[0050] Comparative Example 1

[0051] Take 20 g of barley malt residue after alkali pretreatment, add water to adjust the solid content of the fermentation production system to 10%, add cellulase at an addition amount of 5 FPU / g barley malt residue, and at the same time add lactic acid bacteria seed liquid at an inoculation amount of 10% (v / v). Ferment at 43°C and pH 6.0 for 96 hours to obtain fermentation liquid.

[0052] Comparative Example 2

[0053] (1) Take 20 g of barley malt residue after alkali pretreatment, add water to adjust the solid content of the fermentation production system to 10%, add cellulase at an addition amount of 15 FPU / g barley malt residue, and hydrolyze at 50°C and pH 5.0 for 96 hours to obtain a hydrolyzate;

[0054] (2) Adding the lactic acid bacteria seed solution at an inoculum amount of 10% (v / v) to the hydrolyzate of step (1), and fermenting at 43° C. and pH 6.0 for 96 hours to obtain a lactic acid fermentation solution.

[0055] The concentration changes of lactic acid and acetic acid in the fermentation system in Examples 1-3 and Comparative Examples 1-2, the lactic acid production rate, the lactic acid production per unit of enzyme preparation added, and the lactic acid conversion rate of the fermentable sugar substrate were measured. Figure 1-5 shown.

[0056] In the above embodiments, the selection of specific disclosed data and the selection of any raw materials are not used to limit the scope of protection in the claims. The technical solution of the present invention can be fully implemented by selecting any specific numerical value, raw material, etc. in each numerical range of the claims. For ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention, and these improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for producing lactic acid by fermenting barley malt residue, comprising the following steps: (1) adding water to the alkali-pretreated barley malt residue to adjust the solid content of the fermentation production system to 10%-15%, adding cellulase at an addition amount of 5-10 FPU / g barley malt residue, and hydrolyzing at 50°C-60°C and pH 4.5-5.2 for 20-48 hours to obtain a prehydrolyzed substrate; (2) The hydrolyzate obtained in step (1) was inoculated with a live bacterial count of 1×10 8 -1×10 10 CFU / mL of lactic acid bacteria seed solution, fermenting at 40-55°C and pH 6.0-7.2 for 72-96 hours to obtain a fermentation solution; In the step (1), the preparation method of the barley malt residue after alkali pretreatment is to add alkali solution with a concentration of 0.5 wt%-3.0 wt% to the barley malt residue at a solid-liquid weight ratio of 1:5-1:10, cook at 100°C-120°C for 10-20 min, separate the solid and liquid, and wash the solid residue with water until it is neutral, thereby obtaining the product; In the step (2), the method for preparing the lactic acid bacteria seed liquid is to transfer the frozen glycerol stock of Enterococcus mundtii CGMCC 22227 into MRS medium, incubate for 24-48 hours to obtain an activated seed liquid, and then transfer the activated seed liquid into mMRS medium at an inoculation amount of 10%-15%, and anaerobically culture at 40°C-45°C for 8-12 hours to obtain a viable bacterial count of 1×10 8 -1×10 10 CFU / mL of lactic acid bacteria seed solution.

2. The method according to claim 1, wherein the barley malt residue is a waste biomass raw material generated by using barley in an industrial production process, including residues from industrial brewing, beverage processing, and food production processes and processing residues thereof.

3. The method according to claim 1, wherein the cellulose content of the barley malt residue after the alkali pretreatment is 15%-30% of the barley malt residue dry basis, and the hemicellulose content is 20%-40% of the barley malt residue dry basis.

4. The method according to claim 1, wherein the alkali solution is any one of NaOH and KOH or a combination thereof.

5. The method according to claim 1, wherein the composition of the mMRS medium is: 10 g peptone, 8 g beer extract, 5 g CH3COONa·3H2O, 4 g yeast extract, 2 g K2HPO4, 2 g ammonium citrate, 0.2 g MgSO4·7H2O, 0.05 g MnSO4·4H2O, 1 mL Tween 80, 10 g cellobiose and 10 g xylose per liter of deionized water, the initial pH value is adjusted to 7.0, and sterilized at 115°C for 15 min.

6. The method according to any one of claims 1 to 5, wherein in step (2), the fermentation temperature is 40°C-45°C.

7. The method according to any one of claims 1 to 5, wherein the lactic acid concentration in the fermentation broth is not less than 45 g / L.

8. The method according to claim 7, wherein the lactic acid concentration in the fermentation broth is not less than 50 g / L.

9. The method according to any one of claims 1 to 5, wherein the lactic acid conversion rate of the fermentable sugar substrate is not less than 0.60 g / g.

10. The method according to claim 9, wherein the lactic acid conversion rate of the fermentable sugar substrate is not less than 0.7 g / g.

11. The method according to any one of claims 1 to 5, wherein the lactic acid production rate is not less than 3.0 g / h / L.

12. The method of claim 11, wherein the lactic acid production rate is not less than 3.5 g / h / L.

13. The method according to any one of claims 1 to 5, wherein the concentration of acetic acid in the fermentation broth is less than 2.0 g / L.

14. The method of claim 13, wherein the acetic acid concentration in the fermentation broth is less than 1.3 g / L.

15. The method according to any one of claims 1 to 5, wherein the lactic acid production per unit of enzyme preparation added is greater than 9 (g-Lac / FPU).

16. The method of claim 15, wherein the lactic acid production per unit of enzyme preparation added is greater than 10 (g-Lac / FPU).

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