A kind of efficient lactic acid bacteria and its application in producing lactic acid from kitchen waste

Abstract

The invention relates to a strain of efficient lactic acid-producing bacteria, which is classified and named as Enterococcus faecium, with a Latin name of Enterococcus faecium and a biological collection number of CGMCC No. 22226, and its application in producing lactic acid from kitchen waste. The efficient lactic acid bacteria of the invention are applied to producing lactic acid from kitchen waste, which has the beneficial effects of short fermentation time, high lactic acid production rate, high lactic acid conversion rate of fermentable sugar, high lactic acid accumulation concentration, less byproducts, less carbon dioxide emissions, and the like.

Description

Technical Field

[0001] The invention relates to the field of microbial fermentation, and in particular to a high-efficiency lactic acid-producing bacterium and an application thereof in producing lactic acid from kitchen waste. Background Art

[0002] Lactic acid is a natural organic acid that is widely used in food, pharmaceuticals, chemicals and other fields. It is a bulk liquid chemical required for industrial production. At present, the production of industrial lactic acid is mainly based on fermentation, with grain crops as raw materials, which is costly, and the fermentation production process relies on strict aseptic operation and pure bacterial culture and other technical means. Food waste is rich in nutrients, especially starch-based carbohydrates, which have a content of more than 60% (dry basis), and can be used as a cheap renewable biomass raw material for lactic acid fermentation production. Studies have shown that food waste can be fermented with lactic acid under open conditions without the addition of other nutrients, but the concentration of lactic acid produced is not high, and the lactic acid fermentation liquid obtained contains a variety of by-products, such as ethanol and acetic acid, which leads to high downstream lactic acid purification costs, making it difficult to achieve industrial-scale development of lactic acid production from food waste, and the organic carbon conversion rate is low during the lactic acid production process, and carbon dioxide is easily generated, resulting in carbon source loss. Therefore, it is necessary to study efficient lactic acid bacteria to improve the efficiency of lactic acid production from food waste. Summary of the invention

[0003] The purpose of the present invention is to provide a strain of efficient lactic acid producing bacteria, which is classified and named as Enterococcus faecium, and its Latin name is Enterococcus faecium , the biological deposit number is CGMCC No.22226.

[0004] Another object of the present invention is to provide a method for producing lactic acid from kitchen waste using efficient lactic acid producing bacteria, comprising the following steps:

[0005] (1) Cooking kitchen waste with a moisture content of 70% to 90% in a water bath at 75°C to 95°C for 60 to 120 minutes, and then feeding the waste into a three-phase separator to obtain an oil phase, a solid phase, and a liquid phase. After removing the oil phase, the solid phase and the liquid phase are stirred and mixed at 100 to 150 rpm for 30 to 90 minutes, and then cooled to 55°C to 70°C to obtain a mixed material;

[0006] (2) adding saccharifying enzyme to the mixture prepared in step (1) at an amount of 100-250 U / g of the mixture, stirring continuously at 100-150 rpm for 4-8 h, grinding for 0.5-1 h, and filtering through a 1-2 mm mesh to obtain a saccharification slurry, which is the fermentation substrate;

[0007] (3) Inoculate the fermentation substrate prepared in step (2) with a lactic acid-producing bacteria fermentation seed liquid at an inoculum amount of 5%-15% (v / v), control the fermentation temperature at 35°C-45°C, the pH value at 5.5-7.2, and perform anaerobic fermentation for 2-5 days without sterilization to obtain a fermentation liquid containing lactic acid.

[0008] In a preferred technical solution of the present invention, in step (3), no exogenous nutrients are added during the fermentation process.

[0009] In a preferred technical solution of the present invention, in the step (3), the fermentation pH value is controlled to be between 6.0 and 6.8.

[0010] In the preferred technical solution of the present invention, the food waste refers to the waste obtained after sorting, removing impurities and crushing the food leftovers from restaurants, hotels and company canteens.

[0011] In the preferred technical solution of the present invention, any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonia water or a combination thereof is added to the fermentation system to adjust the pH value.

[0012] In the preferred technical scheme of the present invention, the preparation method of the lactic acid bacteria fermentation seed liquid is as follows: (1) the bacteria preserved in glycerol at -80°C are inoculated into an MRS culture medium containing 20-30 g / L of glucose, and activated and cultured at 35°C-45°C for 9-24 h under anaerobic conditions to obtain an activated seed liquid; (2) the activated seed liquid is inoculated into a new MRS culture medium containing 20-30 g / L of glucose at an inoculation rate of 5-10% (v / v), and expanded and cultured at 35°C-45°C for 8-12 h under anaerobic conditions to obtain a fermentation seed liquid.

[0013] In the preferred technical solution of the present invention, the lactic acid-producing bacteria is Enterococcus faecium, whose Latin name is Enterococcus faecium , the biological deposit number is CGMCC No.22226.

[0014] In the preferred technical scheme of the present invention, the MRS culture medium is: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0-30.0 g of glucose, 5.0 g of sodium acetate trihydrate, 2.0 g of triammonium citrate, 1.0 mL of Tween80, 2.0 g of K2HPO4, 0.2 g of MgSO47H2O, 0.05 g of MnSO4H2O are added to each liter of deionized water, and the pH is 7.0.

[0015] In the preferred technical solution of the present invention, the saccharifying enzyme used is commercially available.

[0016] In a preferred technical solution of the present invention, the pH value of step (2) is maintained within the optimal pH range of the saccharifying enzyme, preferably 4-6.

[0017] In a preferred technical solution of the present invention, the final concentration of lactic acid in the fermentation broth is greater than 110 g / L, preferably greater than 120 g / L.

[0018] In a preferred technical solution of the present invention, the maximum lactic acid production rate in the fermentation broth is greater than 5 g / (L·h), preferably greater than 6 g / (L·h).

[0019] In a preferred technical solution of the present invention, the conversion rate of fermentable sugar lactic acid in the fermentation broth is greater than 85%, preferably greater than 90%.

[0020] In a preferred technical solution of the present invention, the concentration of fermentation by-product acetic acid in the fermentation broth is less than 10 g / L, preferably less than 7 g / L.

[0021] In a preferred technical solution of the present invention, the concentration of fermentation by-product ethanol in the fermentation broth is less than 10 g / L, preferably less than 7 g / L.

[0022] In a preferred technical solution of the present invention, during the fermentation process, the carbon dioxide production is less than 10 mL / 100 g of food waste, preferably less than 5 ml / 100 g of food waste.

[0023] 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.

[0024] Unless otherwise specified, the present invention adopts the following method for detection:

[0025] (1) Test method for lactic acid, acetic acid and ethanol concentration: The sample to be tested was centrifuged in a high-speed centrifuge at 14000 r / min for 5 min, and the supernatant was filtered through a 0.22 μm filter membrane and then entered the liquid chromatography. The sample was separated using a Shimadzu liquid chromatography LC-20A equipped with a ShodexSugar SH1011 liquid chromatography column (8.0 mm×300 mm), and then entered 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.

[0026] (2) Test method for total sugar concentration: Centrifuge the sample at 12000 rpm / min for 10 min, take the supernatant, and dilute the supernatant with distilled water to an appropriate multiple. Take 1 mL of the diluted sample and 2 mL of DNS reagent and mix them in a 25 mL stoppered test tube. Boil in water for 10 min, cool with ice water, and then add distilled water to 25 mL. Measure the absorbance at a wavelength of 550 nm, and substitute the absorbance into the standard curve to calculate the total sugar concentration of the sample.

[0027] (3) Carbon dioxide production test method: The gas production during the fermentation process was measured using a trace gas tester (MGC-1, Ritter, Germany). The carbon dioxide component content was analyzed using a Shimadzu gas chromatograph GC2010plus equipped with a ShinCarbon ST Micropacked Column (19043; 1.00 m × 0.53 mm, 80-100 mesh; Restek; PA, USA) and a TCD detector. Argon was used as the carrier gas, and the temperatures of the column oven, detector, and injection vaporizer were 190°C, 200°C, and 190°C, respectively.

[0028] (4) Conversion rate of fermentable sugar to lactic acid = final concentration of lactic acid in fermentation broth / initial total sugar concentration in fermentation substrate × 100%.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] 1. The present invention provides a highly efficient lactic acid producing bacterium Enterococcus faecium Enterococcus faecium (CGMCC No. 22226), which can tolerate high sugar concentration of fermentation substrate and high lactic acid concentration in fermentation broth, can effectively avoid the inhibition of microbial growth by high concentration substrate and product during fermentation, and is suitable for high concentration lactic acid fermentation production of food waste fermentation substrate with high sugar concentration. At the same time, it can realize efficient and directional bioconversion of carbon source of food waste substrate, and improve lactic acid yield and organic carbon conversion rate.

[0031] 2. The present invention utilizes efficient lactic acid-producing bacteria to produce lactic acid from food waste. Compared with the traditional open fermentation of food waste, it has the advantages of short fermentation time, high lactic acid production rate, high conversion rate of fermentable sugar to lactic acid, high final lactic acid concentration, low by-products, and more than 90% reduction in carbon dioxide emissions.

[0032] The lactic acid bacteria used in the present invention are Enterococcus faecium Enterococcus faecium (CGMCC No.22226), the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation address is No. 3, Yard 1, Beichen West Road, Chanyang District, Beijing, and the preservation date is April 22, 2021. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Comparison of the final concentration of lactic acid in the fermentation broth of Examples 1-3 and Comparative Example 1;

[0034] Figure 2 Comparison of the maximum fermentation rate of lactic acid in the fermentation broth of Examples 1-3 and Comparative Example 1;

[0035] Figure 3 Comparison of the conversion rate of fermentable sugar lactic acid in the fermentation broth of Examples 1-3 and Comparative Example 1;

[0036] Figure 4 Comparison of the yields of byproduct ethanol and acetic acid in the fermentation broth of Examples 1-3 and Comparative Example 1. DETAILED DESCRIPTION

[0037] The following examples are used to illustrate the present invention, but they are not intended to limit the scope of the present invention.

[0038] The food waste from the canteen of Beijing University of Science and Technology was sorted, large pieces of debris were removed, crushed, and the moisture content was adjusted to 75%, which was the food waste in the embodiment of the present invention. The components (% dry weight) in the food waste were detected as follows: starch 54.7%; fat 14.8%; glucose, 1.20%; total sugar 65.5%. The saccharifying enzyme was purchased commercially.

[0039] The lactic acid bacteria used are Enterococcus faecium CGMCC 22226. The steps of culturing the lactic acid bacteria fermentation seed liquid are as follows:

[0040] (1) The bacterial strain preserved in glycerol at -80°C was inoculated into MRS medium containing 20-30 g / L glucose, and activated and cultured at 40°C for 9-24 h under anaerobic conditions to obtain activated seed solution;

[0041] (2) The activated seed liquid is inoculated at a 10% rate into a new MRS medium containing 20-30 g / L of glucose. Under anaerobic conditions, the culture is expanded at 40°C for 8-12 h to obtain the fermentation seed liquid.

[0042] MRS culture medium was as follows: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0-30.0 g of glucose, 5.0 g of sodium acetate trihydrate, 2.0 g of triammonium citrate, 1.0 mL of Tween 80, 2.0 g of K2HPO4, 0.2 g of MgSO47H2O, and 0.05 g of MnSO4H2O were added to each liter of deionized water, and the pH value was 7.0. Example 1

[0043] (1) Take 100 g of kitchen waste, adjust the moisture content to 85%, cook it in a water bath at 80 °C for 90 min, and then send it into a three-phase separator to separate the oil phase, solid phase and liquid phase. After removing the oil phase, stir and mix the solid phase and liquid phase at 120 rpm for 30 min, cool it to 70 °C, and obtain a mixed material;

[0044] (2) Add saccharifying enzyme to the mixture at a rate of 200 U / g of the mixture, stir continuously at 150 rpm for 6 h, grind for 0.5 h in a screw extruder pulper, and filter through a 2 mm sieve to obtain a saccharification slurry, which is the fermentation substrate;

[0045] (3) The fermentation substrate was added to the fermentation tank, and the lactic acid bacteria fermentation seed liquid was inoculated at an inoculation rate of 10%. The pH value of the entire fermentation process was controlled to be maintained at 6.8, the fermentation temperature was 43°C, the fermentation tank was not sterilized, and no exogenous nutrients were added. The anaerobic fermentation was terminated after 72 hours. The final lactic acid concentration, the maximum lactic acid production rate, the fermentable sugar lactic acid conversion rate, and the fermentation byproducts acetic acid and ethanol were detected. Figure 1-4 The total amount of carbon dioxide produced during the entire fermentation process was detected to be 3.4 (mL). Example 2

[0046] (1) Take 100 g of kitchen waste, adjust the moisture content to 80%, cook it in a water bath at 75°C for 60 min, and then send it into a three-phase separator to separate the oil phase, solid phase and liquid phase. After removing the oil phase, stir and mix the solid phase and liquid phase at 150 rpm for 45 min, cool it to 65°C, and obtain a mixed material;

[0047] (2) Add saccharifying enzyme to the mixture at a rate of 150 U / g of the mixture, stir continuously at 120 rpm for 8 h, grind for 0.5 h in a screw extruder pulper, and filter through a 2 mm sieve to obtain a saccharification slurry, which is the fermentation substrate;

[0048] (3) The fermentation substrate was added to the fermentation tank, and the lactic acid bacteria fermentation seed liquid was inoculated at an inoculum rate of 15%. The fermentation pH value was controlled at 7.2 and the fermentation temperature was 45°C. No sterilization was performed and no exogenous nutrients were added. The anaerobic fermentation was terminated after 96 hours. The final lactic acid concentration, the maximum lactic acid production rate, the fermentable sugar lactic acid conversion rate, and the fermentation byproducts acetic acid and ethanol were detected. Figure 1-4 The total amount of carbon dioxide produced during the entire fermentation process was detected to be 3.8 (mL). Example 3

[0049] (1) Take 100 g of kitchen waste, adjust the moisture content to 75%, cook it in a water bath at 95°C for 120 min, and then send it into a three-phase separator to separate the oil phase, solid phase and liquid phase. After removing the oil phase, stir and mix the solid phase and liquid phase at 150 rpm for 60 min, cool it to 60°C, and obtain a mixed material;

[0050] (2) Add saccharifying enzyme to the mixture at a rate of 100 U / g of the mixture, stir continuously at 150 rpm for 4 h, grind for 1 h on a screw extruder pulper, and filter through a 2 mm mesh to obtain a saccharification slurry, which is the fermentation substrate;

[0051] (3) The fermentation substrate was added to the fermentation tank, and the lactic acid bacteria fermentation seed liquid was inoculated at an inoculum rate of 5%. The fermentation pH was controlled at 5.5 and the fermentation temperature was 35°C. No sterilization was performed and no exogenous nutrients were added. The anaerobic fermentation was terminated after 96 hours. The final lactic acid concentration, the maximum lactic acid production rate, the fermentable sugar lactic acid conversion rate, and the fermentation byproducts acetic acid and ethanol were detected. Figure 1-4 The total amount of carbon dioxide produced during the entire fermentation process was detected to be 3.6 (mL).

[0052] Comparative Example 1

[0053] (1) Take 100 g of kitchen waste, adjust the moisture content to 85%, and cook it in a water bath at 80 °C for 90 min. Then, send it into a three-phase separator to separate the oil phase, solid phase and liquid phase. After removing the oil phase, stir and mix the solid phase and liquid phase at 120 rpm for 30 min, cool it to 70 °C, and obtain a mixed material.

[0054] (2) Add saccharifying enzyme to the mixture at a rate of 200 U / g of the mixture, stir continuously at 120 rpm for 6 h, grind for 0.5 h on a screw extruder pulper, and filter through a 2 mm mesh to obtain a saccharification slurry, which is the fermentation substrate;

[0055] (3) The fermentation substrate was added to the fermentation tank, no exogenous lactic acid bacteria were inoculated, the fermentation pH was not controlled, the fermentation temperature was 30°C, no sterilization was performed, no exogenous nutrients were added, and the anaerobic fermentation was terminated after 120 hours. The final lactic acid concentration, the maximum lactic acid production rate, the lactic acid conversion rate of fermentable sugars, and the fermentation byproducts such as acetic acid and ethanol were detected in the fermentation system. Figure 1-4 The total amount of carbon dioxide produced during the entire fermentation process was detected to be 5728.4 mL.

[0056] The above description of the specific embodiments of the present invention does not limit the present invention. Those skilled in the art may make various changes or modifications based on the present invention. As long as they do not depart from the spirit of the present invention, they should all fall within the scope of protection of the claims of the present invention.

Claims

1. A strain of efficient lactic acid bacteria, classified and named: Enterococcus faecium, Latin name Enterococcus faecium , the biological deposit number is CGMCC No.22226.

2. A method for producing lactic acid from kitchen waste using the efficient lactic acid producing bacteria as claimed in claim 1, comprising the following steps: (1) Cooking kitchen waste with a moisture content of 70% to 90% in a water bath at 75°C to 95°C for 60 to 120 minutes, and then feeding the waste into a three-phase separator to obtain an oil phase, a solid phase, and a liquid phase. After removing the oil phase, the solid phase and the liquid phase are stirred and mixed at 100 to 150 rpm for 30 to 90 minutes, and then cooled to 55°C to 70°C to obtain a mixed material; (2) adding saccharifying enzyme to the mixture prepared in step (1) at an amount of 100-250 U / g of the mixture, stirring continuously at 100-150 rpm for 4-8 h, grinding for 0.5-1 h, and filtering through a 1-2 mm mesh to obtain a saccharification slurry, which is the fermentation substrate; (3) Inoculating the fermentation substrate prepared in step (2) with a lactic acid-producing bacteria fermentation seed liquid at an inoculum amount of 5%-15% (v / v), controlling the fermentation temperature at 35°C-45°C and the pH value at 5.5-7.2, and performing anaerobic fermentation for 2-5 days without sterilization to obtain a fermentation liquid containing lactic acid; The lactic acid bacteria are classified and named as Enterococcus faecium, and the Latin name is Enterococcus faecium , the biological deposit number is CGMCC No.22226.

3. The method according to claim 2, characterized in that In step (3), no exogenous nutrients are added during the fermentation process.

4. The method according to claim 2, characterized in that In the step (3), the fermentation pH is controlled at 6.0 to 6.

8.

5. The method according to claim 2, characterized in that The food waste refers to the waste obtained after sorting, removing impurities and crushing the food leftovers from restaurants, hotels and company canteens.

6. The method according to claim 4, characterized in that Any one of sodium hydroxide, potassium hydroxide, calcium hydroxide, sodium dihydrogen phosphate, potassium dihydrogen phosphate, ammonia water or a combination thereof is added to the fermentation system to adjust the pH value.

7. The method according to any one of claims 2 to 6, characterized in that: The preparation method of the lactic acid bacteria fermentation seed liquid is as follows: (1) inoculating the lactic acid bacteria stored in glycerol at -80°C into an MRS culture medium containing 20-30 g / L of glucose, and activating and culturing the culture medium at 35°C-45°C for 9-24 h under anaerobic conditions to obtain an activated seed liquid; (2) inoculating the activated seed liquid into a new MRS culture medium containing 20-30 g / L of glucose at an inoculation rate of 5-10% (v / v), and anaerobic conditions, and expanding and culturing the culture medium at 35°C-45°C for 8-12 h to obtain a fermentation seed liquid.

8. The method according to claim 7, characterized in that The MRS culture medium is as follows: 10.0 g of peptone, 8.0 g of beef extract, 4.0 g of yeast powder, 20.0-30.0 g of glucose, 5.0 g of sodium acetate trihydrate, 2.0 g of triammonium citrate, 1.0 mL of Tween 80, 2.0 g of K2HPO4, 0.2 g of MgSO47H2O, and 0.05 g of MnSO4H2O are added to each liter of deionized water, and the pH value is 7.

0.

9. The method according to any one of claims 2 to 6, characterized in that: The pH value of step (2) is 4-6.

Images