阅读说明：本技术 一种提高卷枝毛霉wj11中链脂肪酸产量的方法 (Method for increasing yield of medium-chain fatty acid of mucor circinelloides WJ11 ) 是由 张怀渊 高萌 宋元达 王瑞雪 赵燕磊 辛菲菲 于 2020-08-04 设计创作，主要内容包括：本发明是一种促进卷枝毛霉WJ11生产中链脂肪酸的方法,属于微生物油脂领域。本发明利用在发酵培养基中添加氯化钴溶液,改变了卷枝毛霉的油脂组成,使其中链脂肪酸的占比增加,发酵条件为：添加氯化钴的终浓度为2 mmol/L,添加时间为接种后的24 h,接入种子液的接种量为10%,发酵罐转速为700 rpm,pH为6.2,空气流速为1 vvm,发酵温度为28℃,发酵时间为120 h,最终中链脂肪酸的含量从1.31%提高到了15.29%,其产量为1109.74 mg/L。(The invention discloses a method for promoting mucor circinelloides WJ11 to produce medium-chain fatty acid, and belongs to the field of microbial oil. The invention changes the grease composition of mucor circinelloides by adding the cobalt chloride solution into the fermentation medium, increases the proportion of the fatty acid therein, and the fermentation conditions are as follows: the final concentration of the added cobalt chloride is 2mmol/L, the adding time is 24 hours after inoculation, the inoculation amount of the inoculated seed liquid is 10 percent, the rotating speed of a fermentation tank is 700 rpm, the pH value is 6.2, the air flow rate is 1vvm, the fermentation temperature is 28 ℃, the fermentation time is 120 hours, the content of the medium-chain fatty acid is improved to 15.29 percent from 1.31 percent, and the yield is 1109.74 mg/L.)

1. A method for improving the yield of medium-chain fatty acid of Mucor circinelloides WJ11 is characterized in that a cobalt chloride solution is added into a culture medium, and the method comprises the following steps: (1) seed culture: inoculating mucor circinelloides WJ11 stored in glycerol at-80 deg.C into seed culture medium, and culturing in constant temperature shaking table at 150 rpm and 28 deg.C for 24 hr; (2) fermentation culture: fermenting in a fermentation tank, inoculating the cultured seed solution into a fermentation culture medium, wherein the inoculation amount is 10%, the final concentration of the added cobalt chloride is 2mmol/L, the addition time is 24h after inoculation, the rotating speed of a stirring paddle of the fermentation tank is 700 rpm, the pH value is 6.2, the air flow rate is 1vvm, the fermentation temperature is 28 ℃, and the fermentation time is 120 h.

2. In claim 1, the seed medium is characterized by: glucose 30 g/L, MgSO₄·7H₂O1.5 g/L, ammonium tartrate 3.3g/L, KH₂PO4 7 g/L，Na₂HPO 42 g/L, yeast extract 1.5 g/L, CaCl₂·2H₂O 0.1 g/L，FeCl₃·6H₂O 8 mg/L，Co(NO₃)₂·6H₂O 0.1 mg/L，ZnSO₄·7H₂O 1 mg/L，CuSO₄·5H₂O 0.1 mg/L，MnSO₄·5H₂O0.1 mg/L; the fermentation medium is characterized in that: glucose 80 g/L, MgSO₄·7H₂O1.5 g/L, ammonium tartrate 2.0 g/L, KH₂PO4 7 g/L，Na₂HPO 42 g/L, yeast extract 1.5 g/L, CaCl₂·2H₂O 0.1 g/L，FeCl₃·6H₂O 8 mg/L，Co(NO₃)₂·6H₂O 0.1 mg/L，ZnSO₄·7H₂O 1 mg/L，CuSO₄·5H₂O 0.1mg/L， MnSO₄·5H₂O 0.1 mg/L。

3. The process according to claim 1, characterized in that the final concentration of cobalt chloride added to the culture medium is 2mmol/L and the time of addition to the culture medium is 24h after inoculation.

Technical Field

The invention belongs to the field of microbial oil. The fermentation method can promote mucor circinelloides WJ11 to produce medium-chain fatty acids, and the yield is considerable.

Background

The filamentous fungi widely occur in nature, Mucor circinelloides (Mucor circinelloides) Is one of the commonThe strain with high oil yield can produce a large amount of linoleic acid and gamma-linolenic acid and accumulate in cells. The strain used in the invention is mucor circinelloides WJ11, which can produce lipid accounting for 36% of dry cell weight (CDW), but the lipid is long-chain fatty acid with more than C14, such as gamma-linolenic acid (cis-6, 9, 12-octadecatrienoic acid) and the like.

Medium-chain fatty acids (MCFA) are a general term for fatty acids of a certain chain length, and generally refer to fatty acids having six to twelve carbon chains such as caprylic acid and capric acid. Medium chain fatty acids are important precursors for food, medical and chemical products. The ingested medium-chain fatty acid is directly transported to the liver through the portal vein to participate in oxidation, thereby rapidly providing energy, causing no obesity and being very beneficial to extreme athletes and weight-losing people. The medium-chain fatty acid in nature is mainly present in coconut oil and palm kernel oil, but the content is not high (less than 15% of the total oil content). In industrial production, the source of medium-chain fatty acids is mainly artificial chemical synthesis or genetic engineering modification, and the methods for producing medium-chain fatty acids by oil-producing microorganisms are also few.

Disclosure of Invention

The invention develops a convenient and effective method for improving the yield of medium-chain fatty acid, namely adding a cobalt chloride solution into a fermentation medium. Compared with artificial chemical synthesis and genetic engineering operation, the method is time-saving and labor-saving, and has considerable benefits.

The technical scheme is as follows:

in previous experiments, we selected Al³⁺、Fe²⁺、Co²⁺、Ni²⁺、Cu²⁺Adding various metal ions into a culture medium of Mucor circinelloides WJ11 respectively, measuring the content of grease generated by Mucor circinelloides WJ11 and finding out Co²⁺The addition of (2) makes Mucor circinelloides WJ11 produce medium chain fatty acids C10 and C12. We have conducted intensive studies on this phenomenon by controlling Co²⁺Concentration of (C) and Co²⁺The addition time of (a) is different to determine the optimal fermentation conditions.

The invention has the following benefits and effects:

Co²⁺the addition of the compound can promote the synthesis of medium-chain fatty acid of the mucor circinelloides WJ11,the proportion is increased, and the optimal fermentation condition is selected by experiment, namely 2mmol/L Co is added in 24h²⁺Under the fermentation conditions Co²⁺The effect of promoting the synthesis of medium-chain fatty acid of the mucor circinelloides is best, and the medium-chain fatty acid accounting for 15.3 percent of the total grease content can be generated by the mucor circinelloides, and the content of the medium-chain fatty acid can reach 1109.7 mg/L.

Detailed Description

Strain:

mucor circinelloides WJ11 (Mucor circinelloidesWJ11, CCTCC No. M 2014424; China Centerfor Type Culture Collection）

Optimum Co²⁺Determination of concentration:

the invention designs 6 different groups of Co²⁺The concentration gradients are added into 6 identical baffle triangular flasks respectively when the culture medium is prepared, each triangular flask is filled with 200 mL of fermentation medium, the formula is 80 g/L glucose and MgSO₄·7H₂O1.5 g/L, ammonium tartrate 2.0 g/L, KH₂PO4 7 g/L，Na₂HPO 42 g/L, yeast extract 1.5 g/L, CaCl₂·2H₂O 0.1 g/L，FeCl₃·6H₂O 8 mg/L，Co(NO₃)₂·6H₂O 0.1 mg/L，ZnSO₄·7H₂O 1 mg/L，CuSO₄·5H₂O 0.1 mg/L， MnSO₄·5H₂O0.1 mg/L, the inoculum size is 10 percent, and the mixture is put into a constant temperature shaking table for culture, the rotating speed is 150 rpm, the temperature is 28 ℃, and the culture time is 96 hours.

TABLE 1 different Co²⁺Is added in an amount of

Numbering	1	2	3	4	5	6
							Co2+Concentration (mmol/L)	0	1	2	3	4	5
CoCl2Solution (mu L)	0	10	20	30	40	50

TABLE 2 different Co²⁺In an amount of

Co2+Concentration (mmol/L)	0	1	2	3	4
						Medium chain fatty acid content (%)	1.385	4.358	9.022	7.702	7.175

The experimental results show that the sum of C10 and C12, i.e. the content of medium-chain fatty acids, is influenced by Co²⁺Influence of (1), Co²⁺Mucor circinelloides does not grow at the concentration of 6mmol/L, and the Mucor circinelloides does not grow at Co²⁺The maximum value is obtained when the addition amount of (C) is 2mmol/L, and the content of medium-chain fatty acid is the highest, and the medium-chain fatty acid accounts for 9.022% of the total oil content.

Optimum Co²⁺Adding time:

the experimental group comprises four identical baffle triangular flasks, and each triangular flask is filled with 200 mL of fermentation medium and optimal Co²⁺The concentration is 2mmol/L, and the solution is added at the time of solution preparation (0 h), after one day (24 h), after two days (48 h) and after three days (72 h). The inoculation amount is 10 percent, and the mixture is put into a constant temperature shaking table for cultivation at the rotating speed of 150 rpm and the temperature of 28 ℃ for 96 hours.

TABLE 3 different Co²⁺Production of medium chain fatty acids with addition time

Co2+AddingTime (d)	0	1	2	3
					Medium chain fatty acid content (%)	9.917	14.543	5.316	4.202

The experimental results show that when Co is added after one day of fermentation²⁺The content of medium-chain fatty acid in the mucor circinelloides is highest, wherein the medium-chain fatty acid accounts for 14.543% of the total oil content. Adding Co after fermenting for two days²⁺The promotion effect on the generation of the medium-chain fatty acid is reduced, which is lower than that of adding Co when preparing the culture medium²⁺. Thus adding Co after one day of fermentation²⁺Is the optimal choice.

Fermenting in a fermentation tank

Seed culture: inoculating Mucor circinelloides WJ11 stored in glycerol at-80 deg.C into seed culture medium, culturing in constant temperature shaking table at 150 rpm and 28 deg.C for 24 hr to obtain seed solution.

Four fermentation tanks are adopted for simultaneous fermentation in the experiment, the numbers of the four fermentation tanks are F1, F2, F3 and F4 in sequence, the set fermentation conditions are shown in table 2, the rotating speed of the fermentation tanks is 700 rpm, the pH value is 6.2, the temperature is 28 ℃, the air flow rate is 1vvm, and the fermentation time is 120 h.

TABLE 4 fermentation conditions in the fermenter

Numbering	F1	F2	F3	F4
					Seed liquid (mL)	100	100	100	100
Lipid production medium (mL)	900	900	900	900
					Co2+Concentration (mmol/L)	0	2	2	1
Co2+Addition time (h)	0	0	24	24

TABLE 5 fermenter 120h fatty acid composition (%)

	F1	F2	F3	F4
					C10:0	0.34±0.05	4.27±0.37	4.67±0.36	4.23±0.37
C12:0	0.97±0.11	9.28±0.91	10.62±0.56	9.04±0.89
					C14:0	2.05±0.95	7.20±0.85	7.09±0.74	6.15±0.62
C16:0	33.29±1.2	20.02±1.17	21.61±1.58	21.55±1.57
					C18:0	6.23±0.85	34.56±1.45	34.97±1.76	31.96±1.38
C18:1	31.56±1.87	8.51±0.95	7.96±0.82	9.81±1.01
					C18:2	12.35±1.21	10.28±1.05	8.76±0.88	10.53±1.07
C18:3	13.21±1.35	5.88±0.36	4.33±0.45	6.74±0.72

TABLE 6 content of fatty acids in fermenter (%)

	F1	F2	F3	F4
					24h	0.191	1.35	0.236	0.973
48h	0.714	7.573	0.809	6.949
					72h	1.132	10.683	9.275	8.73
96h	1.205	12.734	13.859	11.295
					120h	1.31	13.553	15.29	13.267

TABLE 7 yield of medium chain fatty acids (mg/L) in fermentors

	F1	F2	F3	F4
					24h	0.700	1.545	1.054	1.228
48h	29.700	89.615	36.052	186.447
					72h	68.762	375.340	505.000	421.404
96h	94.201	596.570	969.578	757.936
					120h	108.071	680.118	1109.737	902.018

As can be seen from the results of the experiments, Co was added to the medium²⁺The fatty acid composition of Mucor circinelloides is influenced, the content of C10:0, C12:0, C14:0 and C18:0 is obviously improved relative to the control group, and 2 mmol/LCo is added when 1 day is added²⁺When the content of the medium-chain fatty acid in the mucor circinelloides grease is highest, the content of the medium-chain fatty acid in the mucor circinelloides grease is highest. At the end of fermentation, 2 mmol/LCo was added at 1 day²⁺Under the condition, the yield of medium-chain fatty acid produced by the mucor circinelloides is 1109.7 mg/L.