CN102732575A - Method for producing L-lactic acid through fermentation of rhizopus oryzae - Google Patents

Abstract

The invention provides a method for producing L-lactic acid through the fermentation of rhizopus oryzae. The method is characterized by taking rhizopus oryzae as a starting strain to produce L-lactic acid through fermentation. The fermentation process conditions for producing L-lactic are determined by the method.

Description

A kind of method of utilizing the Rhizopus oryzae fermentation production of L-lactic acid

Technical field

The invention belongs to fermentation technical field, be specifically related to a kind of method of utilizing the Rhizopus oryzae fermentation production of L-lactic acid.

Background technology

L-lactic acid is one of the organic acid of tool application potential of 21 century.L-lactic acid and verivate thereof are important Organic Chemicals, are again important fine chemicals, are widely used in fields such as food, agricultural, environmental protection, medicine, feed, daily necessities, chemical industry.The bacterial classification of fermentative Production L lactic acid mainly contain lactobacillus genus ( Lactobacillus) and Rhizopus ( Rhizopus).Use root arrhizus fermentation to produce L lactic acid and be aerobic fermentation, have shortcomings such as big, the sugared utilization ratio of process energy consumption is low.Adopt lactobacillus-fermented produce L lactic acid have sugared utilization ratio high, not oxygen consumption, energy consumption is low, equipment is simple, advantage such as easy to operate, in lactic acid industry, has vast potential for future development.

Abroad lactic acid fermented research mainly concentrates on and continuously ferments and fermentation and stripping technique coupling mutually about L at present, electrodialysis is combined timely separating lactic acid, control pH value like Li etc. with the bipolar membrane electrodialysis technology.Gao etc. use continuous electric dialysis fermentation device, and L lactic acid yield, yield reach 8.18 g/ (L h) and 0.69 g/g respectively.Continuously ferment and to eliminate the product inhibition, increase substantially the production efficiency of L lactic acid, but it is ripe not enough to be applied to scale operation.The research that domestic employing lactobacillus-fermented is produced L lactic acid is main with batch fermentation, knows clean grade through medium optimization as happy, and the L lactic acid production reaches 103 g/L.Yan Zhihui etc. are with in the ammoniacal liquor and lactic acid; L lactic acid concn, productive rate reach 136.8 g/L and 1.71g/ (L h) respectively. and Li Jian etc. add multiple amino acids and growth factor in substratum; L lactic acid production, yield and productive rate reach 140.3 g/L respectively, 0.94g/g and 1.95 g/ (L h).Application constant speed such as Bai stream adds the fermentation strategy, and L lactic acid production, yield and productive rate reach 210 g/L respectively, 0.97 g/g and 2.2 g/ (L h).More than the deficiency of research existence is the medium component more complicated, comprising expensive raw materials such as yeast powder, peptones.The research of Kwon etc. shows that the cost of nitrogenous source such as yeast powder accounts for about 30% of total fermentation costs in the lactobacillus ferment.

Summary of the invention

The invention provides a kind of method of utilizing the Rhizopus oryzae fermentation production of L-lactic acid, the present invention is starting strain with the Rhizopus oryzae, and through fermentative prodn L lactic acid, the present invention has confirmed to produce the technological condition for fermentation of L-lactic acid.

1. method of utilizing the Rhizopus oryzae fermentation production of L-lactic acid; When it is characterized in that fermenting, a certain amount of substratum of in 250 mL substratum, packing into inserts a certain amount of seed culture medium to fermention medium; Add the lime carbonate of sterilization separately, at 35 ℃ of condition bottom fermentation 72 h.

2. method according to claim 1 is characterized in that, fermention medium (g/L): W-Gum 120, ammonium sulfate 3, potassium primary phosphate 0.25, zinc sulfate 0.04, sal epsom 0.15, pH value 6.0 adds lime carbonate 60 then.

3. method according to claim 1 is characterized in that, the preparation method of seed culture medium is: the seed culture medium sterilization, insert cultured spore, shaking culture 24 h under 32 ℃ of conditions.

4. method according to claim 3 is characterized in that, seed culture medium (g/L): glucose 100, ammonium sulfate 1.35, potassium primary phosphate 0.3, zinc sulfate 0.04, sal epsom 0.25, pH value 6.0.

5. method according to claim 1 is characterized in that, definite employing shake flat experiment of inoculum size and culture temperature, 250 mL shake bottled liquid measure 100 mL.Shaking speed is 150 r/min, and the pH regulator is 2 g CaCO ₃, sterilization separately, aseptic adding when inoculation.

6. method according to claim 2, it is characterized in that W-Gum concentration about 14% for well.

7. method according to claim 1 is characterized in that ammonium sulfate is optimum nitrogen source.

8. method according to claim 1 is characterized in that potassium primary phosphate produces the source of phosphoric acid of L-lactic acid as fermentation.

9. method according to claim 1 is characterized in that best liquid amount 40 mL/250mL.

Beneficial effect of the present invention is: the present invention is starting strain with the Rhizopus oryzae, and through fermentative Production L lactic acid, the present invention has confirmed to produce the technological condition for fermentation of L-lactic acid.

Embodiment

Following embodiment elaborates to the present invention, but to not restriction of the present invention.

The used bacterial strain of the present invention is a Rhizopus oryzae, buys in ACCC, is numbered: ACCC31821

Embodiment 1

This case study on implementation is explained different carbon source concentrations to producing the influence of L-lactic acid, and carbon source concentration is respectively: 10%, 12%, 14%, 16%, 18% and 20%.The result is as shown in table 1.

Can find out that from table 1 along with the increase of W-Gum concentration, product acid amount is increasingly high, but transformation efficiency is more and more lower.When concentration was lower than 14%, though product acid amount is low, raw material was fully used, and transformation efficiency raises; When concentration is higher than 14%, slightly increase though produce the acid amount, transformation efficiency descends bigger.So take all factors into consideration, W-Gum concentration about 14% for well.

The different carbon source concentrations of table 1 are to producing the influence of acid

Carbon source concentration (%)	Total reducing sugar (g/L)	L-lactic acid (g/L)	Residual sugar (g/L)	Transformation efficiency (%)
					10	92.0	78.32	3.24	88.24
12	112.5	84.12	5.24	78.43
					14	132.2	98.34	1.78	75.40
16	149.6	103.43	2.26	70.20
					18	168.8	104.98	4.34	63.94
20	185.0	110.32	5.34	61.40

Embodiment 2

This case study on implementation explanation different nitrogen sources is to producing the influence of L-lactic acid; Nitrogenous source is respectively: urea, an ammonium nitrate, ammonium sulfate, steeping water, soya-bean cake hydrolyzed solution and glutamic acid-fermented waste liquid; Solid nitrogenous source addition is 0.3% (mass ratio), and liquid nitrogenous source addition is 3% (V/V), and the result sees table 2.

By knowing in the table 2, the various nitrogenous sources of Rhizopus oryzae interpolation capable of using produce L-lactic acid, and wherein the product of ammonium sulfate acid amount is the highest, therefore selects ammonium sulfate as fermentation nitrogen source.

Table 2 different nitrogen sources is to producing the influence of L-lactic acid

Nitrogenous source	L-lactic acid (g/L)	Residual sugar (g/L)	Transformation efficiency (%)
				Urea	74.32	9.23	62.34
An ammonium nitrate	104.46	2.30	79.25
				Ammonium sulfate	108.86	3.12	85.54
Steeping water	32.34	14.21	27.45
				The soya-bean cake hydrolyzed solution	63.52	12.46	53.75
Glutamic acid-fermented waste liquid	101.34	2.43	80.23

Embodiment 3

This case study on implementation explanation different phosphate hydrochlorate is the basis with the fermention medium to producing the influence of acid, adopts the fermentation of different phosphate hydrochlorate, and phosphate adding quantity is 0.025%, and the result sees table 3.

Visible from table, several kinds of phosphoric acid salt that try all can be done the phosphorus source of Rhizopus oryzae fermenting L-lactic acid, and wherein the potassium primary phosphate effect is best, produce the acid height, and transformation efficiency is also high, therefore select potassium primary phosphate to produce the source of phosphoric acid of L-lactic acid as fermentation.

Table 3 different phosphate hydrochlorate is to producing the influence of acid

Phosphoric acid salt	L-lactic acid (g/L)	Residual sugar (g/L)	Transformation efficiency (%)
				Potassium primary phosphate	108.65	1.04	85.48
SODIUM PHOSPHATE, MONOBASIC	98.86	2.30	76.25
				Primary ammonium phosphate	102.86	0.12	78.26
Secondary calcium phosphate	100.37	1.87	77.65

Embodiment 4

This case study on implementation is explained the influence of liquid amount to product L-lactic acid, the fermention medium of packing 20,40,60,80 mL in 250 mL triangular flasks, and 72 h that ferment, the result sees table 4.

Can find out that from table 4 liquid amount is higher at 20-40 mL scope transformation efficiency, liquid amount is lower at 60-80 mL scope transformation efficiency.This explanation Rhizopus oryzae carries out the L-lactobacillus ferment and ventilation is required little.Finally choosing inoculum size is 40 mL/250 mL.

Table 4 liquid amount is to producing the influence of L-lactic acid

Liquid amount (mL)	L-lactic acid (g/L)	Residual sugar (g/L)	Transformation efficiency (%)
				20	93.36	2.65	85.68
40	95.16	2.30	86.25
				60	92.82	1.12	81.26
80	90.37	0.87	79.65

Claims (9)

1. method of utilizing the Rhizopus oryzae fermentation production of L-lactic acid; When it is characterized in that fermenting, a certain amount of substratum of in 250 mL substratum, packing into inserts a certain amount of seed culture medium to fermention medium; Add the lime carbonate of sterilization separately, at 35 ℃ of condition bottom fermentation 72 h.

2. method according to claim 1; It is characterized in that; Fermention medium: W-Gum 120 g/L, ammonium sulfate 3 g/L, potassium primary phosphate 0.25 g/L, zinc sulfate 0.04 g/L, sal epsom 0.15 g/L, pH value 6.0 adds lime carbonate 60 g/L then.

3. method according to claim 2 is characterized in that W-Gum concentration 14%.

4. method according to claim 1 is characterized in that, the preparation method of seed culture medium is: the seed culture medium sterilization, insert cultured spore, shaking culture 24 h under 32 ℃ of conditions.

5. method according to claim 4 is characterized in that seed culture medium: glucose 100 g/L, ammonium sulfate 1.35 g/L, potassium primary phosphate 0.3 g/L, zinc sulfate 0.04 g/L, sal epsom 0.25 g/L, pH value 6.0.

6. method according to claim 1 is characterized in that, definite employing shake flat experiment of inoculum size and culture temperature, 250 mL shake bottled liquid measure 100 mL; Shaking speed is 150 r/min, and the pH regulator agent is 2 g CaCO ₃, sterilization separately, aseptic adding when inoculation.

7. method according to claim 1 is characterized in that nitrogenous source is an ammonium sulfate.

8. method according to claim 1 is characterized in that potassium primary phosphate produces the source of phosphoric acid of L-lactic acid as fermentation.

9. method according to claim 1 is characterized in that liquid amount 40 mL/250mL.