CN110295213B - Method for fermentation production of umami peptide based on index flow feeding principle - Google Patents

Method for fermentation production of umami peptide based on index flow feeding principle Download PDF

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CN110295213B
CN110295213B CN201910387109.8A CN201910387109A CN110295213B CN 110295213 B CN110295213 B CN 110295213B CN 201910387109 A CN201910387109 A CN 201910387109A CN 110295213 B CN110295213 B CN 110295213B
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孙伟峰
伍圆明
王莉
伍伦杰
林璐
朱龙宝
王剑锋
关成冉
陈晓华
刘义
车振明
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Abstract

The invention discloses an umami peptide fermentation production method based on an index flow feeding principle. The method is based on a fermentation kinetic model of the umami peptide biological expression bacteria, utilizes the Pontrieau maximum value principle, and solves the optimal process change of the specific growth rate when the maximum value is obtained by the target function within a certain limit condition range by establishing a state equation and the target function, and controls the fermentation production of the umami peptide. The fermentation production optimization method effectively improves the yield of the fresh peptide, and can be widely applied to the field of fermentation production of peptide or protein.

Description

Method for fermentation production of umami peptide based on index flow feeding principle
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a fermentation production method of umami peptide based on an index flow feeding principle.
Background
The delicious peptide is a kind of polypeptide or its salt which is composed of two or more amino acids and can make human body feel delicious at a certain concentration. The beef flavor Peptide (Beefy meet Peptide, BMP) is a flavor Peptide which is widely researched at present, and can participate in Maillard reaction with substances such as reducing sugar, amino acid, nucleotide and the like in addition to the characteristic flavor of the beef flavor Peptide, so that a special compound flavor is generated, the characteristics of coordinated taste, freshness, mellowness and the like of food are endowed, and the beef flavor Peptide can be independently used as a flavor seasoning or used as a base material to prepare a novel compound seasoning, and has great market potential. At present, the reported BMP production modes at home and abroad are divided into 3 types: chemical synthesis, enzymatic decomposition and biological expression, wherein the former two are not suitable for actual production due to the limitations of small yield, high synthesis cost, complex separation and purification and the like, and compared with the production of the umami peptide by biological expression and fermentation, the method has the advantages of high efficiency, rapidness and low cost, and provides feasibility for large-scale production of the umami peptide. However, the research on the biological fermentation production of the delicious peptide BMP is still in the initial stage at present, and compared with the yield of the fermentation production of other peptide products, the yield of the delicious peptide BMP is improved.
The high-density fermentation technology is a common technology for expanding the production of microorganisms, and can control the cell quantity of thalli to be accumulated as much as possible within a certain reactor volume and time, thereby improving the yield of target products. In the high-density fermentation process, a fermentation factor which has a large influence on the growth and production of thalli is substrate inhibition, and the prior substrate inhibition removal is mainly realized by feeding control. For a growth-coupled expression system, because the accumulation of thalli and the expression of products are mainly concentrated in the exponential growth phase of thalli, an exponential flow feeding strategy is often adopted to remove the substrate inhibition. The calculation formula (formula 1) used by the fed-batch strategy is derived from a growth kinetic model, wherein the key parameter is compared with the set value (mu) of the growth rateset) Usually, the method is an experimental empirical value, so that the supplement amount of exponential flow addition is not completely matched with the actual growth requirement of thalli, and finally, the density of the thalli and the yield of products are limited. Therefore, for the exponential fed-batch strategy of high-density fermentation, it is necessary to further optimize mu thereofsetThe substrate concentration is adjusted to satisfy the requirements of bacterial growth and production while simultaneously eliminating substrate inhibition.
Figure BDA0002055206250000021
Wherein F represents the feed addition rate, μsetShowing the specific growth rate of the cells, X0Shows the cell concentration in the fermenter at the start of replenishing the feed, V0Denotes the volume of medium in the fermenter at the start of the feed, SFDenotes the substrate glucose concentration in the feed medium, YX/SThe cell growth is based on the yield coefficient of the substrate, and t represents the feeding time.
The Pontrieau maximum principle is commonly used for solving the optimal control problem in the engineering field, and the specific solving process is as follows: establishing a series of state equations (equation 12) and an objective function (equation 13) for describing the state change of the object, introducing a Lagrange multiplier (lambda (t)) which evolves along with time to establish a Hamiltonian (equation 14), further determining necessary conditions for solving the maximum value of the objective function, namely an adjoint function (equation 15) and a control equation (equation 16), and simultaneously solving the equations 12, 15 and 16 to obtain the optimal solution of the corresponding control variable when the maximum value is obtained by the objective function. The application of the Pontrieau maximum principle to the fermentation process control of the umami peptide is not reported.
Figure BDA0002055206250000022
Figure BDA0002055206250000023
Figure BDA0002055206250000024
Figure BDA0002055206250000025
Figure BDA0002055206250000026
Disclosure of Invention
Aiming at the prior art, the invention provides a fermentation production method of umami peptide based on the principle of exponential flow feeding, and the fermentation production of the umami peptide is improved by the fermentation method.
In order to achieve the purpose, the invention adopts the technical scheme that: provides a fermentation production method of the umami peptide based on the index fed-batch principle, the fed-batch feeding rate in the fermentation process of the umami peptide is determined by the formula (1),
Figure BDA0002055206250000031
wherein F represents the feed addition rate, μsetShowing the specific growth rate of the cells, X0Shows the cell concentration in the fermenter at the start of replenishing the feed, V0Denotes the volume of medium in the fermenter at the start of the feed, SFDenotes the substrate glucose concentration in the feed medium, YX/SThe yield coefficient of the thallus growth based on a substrate, and t represents the feeding time; specific growth rate of thallussetThe method is obtained by optimizing the Pontrieau maximum value principle.
On the basis of the technical scheme, the invention can be further improved as follows.
Further, the specific growth rate of the cells is μsetThe optimization comprises the following steps:
s1: establishing a fermentation kinetic model of the umami peptide biological expression engineering bacteria;
Figure BDA0002055206250000032
Figure BDA0002055206250000033
Figure BDA0002055206250000034
wherein, the formula 2 is a thallus growth rate equation, the formula 3 is a substrate consumption rate equation, and the formula 4 is a product synthesis rate equation; x is the thallus density in the fermenter during the feed-batch fermentation, S is the glucose concentration in the fermenter during the feed-batch fermentation, P is the product concentration in the fermenter during the feed-batch fermentation, YP/SFor the product substrate-based yield coefficient, qmFor a specific substrate consumption rate coefficient for cell maintenance, α is a product expression rate coefficient related to cell growth rate and β is a cell density-related product expression rate coefficient;
s2: determining a state equation, an objective function and a control condition to be solved in the Pontrieau maximum principle based on the fermentation kinetic model of S1;
Figure BDA0002055206250000041
Figure BDA0002055206250000042
Figure BDA0002055206250000043
Figure BDA0002055206250000044
wherein, the equations 5-7 are state equations, and the equation 8 is a target function;
s3: based on the state equation and the objective function in step S2, introducing a lagrange multiplier to construct a hamiltonian, and determining necessary conditions for extremum calculation of the objective function: an adjoint function and a governing equation;
H＝λ1f1+λ2f2+(1+λ3)f 3 9
Figure BDA0002055206250000051
Figure BDA0002055206250000052
wherein, formula 9 is a Hamiltonian, formula 10 is an adjoint function, and formula 11 is a control equation;
s4: simultaneously solving the state equation, the adjoint function and the control equation in the steps S2 and S3, determining the optimal process change of the corresponding control condition when the objective function obtains an extreme value in a given condition range, namely, optimizing to obtain the specific growth rate mu of different time periodsset。
Further, the umami peptide is beef flavor peptide, and the sequence of the umami peptide is shown in SEQ.ID.No. 1.
Further, Bacillus subtilis 168 is taken as a host bacterium, pMA09 is taken as a vector plasmid, and PsrfA with self-induction characteristic and high-efficiency expression is taken as a promoter in the umami peptide biological expression engineering bacterium in S1.
Further, the medium included the following components in the following concentrations: 7.5g/L diammonium hydrogen citrate, 2.0g/L sodium sulfate, 20.0g/L ammonium sulfate, 3.5g/L ammonium chloride, 14.6g/L dipotassium phosphate, 4.0g/L sodium dihydrogen phosphate monohydrate, 1.0g/L magnesium sulfate heptahydrate, 20.0g/L glucose and 3.0mL of trace metal ion solution.
Further, the trace metal ion solution comprises the following components in concentration: 0.5g/L of calcium chloride, 0.18g/L of zinc sulfate heptahydrate, 0.1g/L of manganese sulfate monohydrate, 10.05g/L of disodium ethylene diamine tetraacetate, 8.35g/L of ferric chloride, 0.16g/L of copper sulfate pentahydrate and 0.18g/L of cobalt chloride hexahydrate.
The invention has the beneficial effects that:
compared with the prior art, the fermentation production method of the umami peptide based on the index fed-batch principle can control the index fed-batch rate in real time in multiple stages in the fermentation process, remove the inhibition of the substrate, simultaneously enable the restrictive substrate to effectively match the growth requirement of host bacteria, further increase the biomass of thalli, improve the fermentation strength and the yield (BMP yield is 3.16g/L) of the umami peptide, and realize the high-efficiency production of the umami peptide. The optimized fermentation method has obvious effect, the biomass and the umami peptide yield are respectively 3.2 times and 2.8 times of batch fermentation, and a solid theoretical basis is provided for the large-scale production of BMP and other umami peptides or proteins.
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FIG. 1 is a diagram showing the result of agarose electrophoresis of a recombinant plasmid; m represents DNA marker, 1 represents protoplasmid pMA09-8BMP, and 2 represents recombinant plasmid pMA09srfA-8 BMP;
FIG. 2 is a graph of taste peptide BMP index fed-batch fermentation optimized based on Pontrieau maximum principle; "■" indicates DCW, ". major" indicates the production amount of the umami peptide, ". tangle-solidup" indicates the concentration of glucose, "-" indicates DO;
FIG. 3 is a diagram of batch fermentation of the umami peptide BMP; "□" indicates DCW, "four" indicates umami peptide production, ". DELTA" indicates glucose concentration, "-" indicates DO.
Detailed Description
The present invention will be described in further detail with reference to examples of the present invention, but it should not be construed that the scope of the above-described subject matter of the present invention is limited to the examples. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The first embodiment is as follows: construction of efficient self-induced production strain of umami peptide
The method comprises the steps of taking an umami peptide expression strain B.subtilis 168/pMA09-8BMP (xylol. cloning expression of umami peptide BMP and high-density fermentation production [ D ]. Siwa university, 2018) which are successfully constructed in the prior art as a research object, replacing a promoter in a primary plasmid pMA09 with a high-efficiency self-induction expression type promoter srfA, and constructing a recombinant plasmid for high-efficiency self-induction expression of the umami peptide. The gene sequence of the self-inducible promoter srfA is shown in SEQ ID NO.2, the original plasmid pMA09 is used as a PCR template by using whole plasmid PCR, and the results of the designed homologous arm sequences are shown in Table 1.
TABLE 1 homology arm sequence for pMA09 Whole plasmid PCR
Figure BDA0002055206250000071
A recombinant plasmid pMA09-8BMP in B.subtilis 168/pMA09-8BMP is extracted by an alkaline lysis method, a fresh peptide self-induced expression vector pMA09srfA-8BMP is obtained by a whole plasmid PCR amplification technology, the result of agarose gel electrophoresis verification of the recombinant plasmid is shown in figure 1, a lane 2 is a gene strip of the original plasmid pMA09-8BMP, a gene strip of a lane 3 is larger than a gene strip of the lane 2, the size of the gene strip is 7642bp, the design result is the same as that of the gene strip, and the successful construction of the recombinant plasmid pMA09srfA-8BMP of the self-induced expression promoter is verified by sequencing. The recombinant plasmid is transformed into a blank recombinant strain B.subtilis 168, so as to obtain the high-efficiency self-induced production strain B.subtilis 168/pMA09srfA-8BMP of the umami peptide.
Example two: establishment of fermentation kinetic model of umami peptide biological expression engineering bacteria
The promoter P used in the thallus B.subtilis 168/pMA09srfA-8BMPsrfAThe promoter is a cell density dependent promoter, has certain synchronism in thallus density accumulation and umami peptide expression, and is mainly concentrated in an exponential growth stage of thallus, so that the stage is used as a feeding fermentation control stage. In the fermentation process, glucose concentration is the only substrate limiting condition, and the growth and product expression of the thalli are determined to be suitable for a growth coupled model with substrate inhibition and product inhibition (formulas 17-21) in a feeding stage by combining the expression rule of the promoter:
μ＝qgYX/S 17
qs＝qm+qg+qp 18
Figure BDA0002055206250000081
Figure BDA0002055206250000082
Figure BDA0002055206250000083
the feeding control of the exponential growth phase of the thalli mainly controls the glucose amount in the fermentation tank, so that the glucose amount can meet the growth requirement of the thalli, and meanwhile, the glucose amount cannot be accumulated to generate substrate inhibition on the growth of the thalli, and the feeding at the stage is carried out according to the exponential flow feeding (formula 1). On the basis, combining the formula 17-21, setting mu for the optimization of the subsequent Pontrieau maximum value principle on the condition exploration of the mu valuesetThe fermentation kinetic model of the thallus at the index feeding stage is arranged as follows: a thallus growth rate equation (formula 2), a substrate consumption rate equation (formula 3) and a product synthesis rate equation (formula 4).
Figure BDA0002055206250000084
Figure BDA0002055206250000085
Figure BDA0002055206250000091
In the course of exponential feed supplement,. mu.setThe value has an important role in determining the substrate requirement of the bacterial cells. In the actual fermentation process, the mu of the exponential growth phase of the thallussetThe value is changed continuously, only a fixed mu is setsetThe exponential feeding is not the best control to meet the actual growth requirement of the thalli, so that the mu is reducedsetThe values are used as unknown control variables for the following Pontrieau maximum principle optimization. Initial parameter X of feeding in the fermentation kinetic model0、V0、SFObtained by detection during the actual fermentation (table 2), the remaining kinetic parameters: q. q.sm(h-1)、YX/S(g/g)、YP/S(g/g), α, β are shown in Table 3.
TABLE 2 initial value of exponential feeding during fermentation
Figure BDA0002055206250000092
TABLE 3 values of kinetic parameters during fermentation
Figure BDA0002055206250000093
The parameters are substituted to obtain a specific fermentation kinetic model as follows:
Figure BDA0002055206250000094
Figure BDA0002055206250000095
Figure BDA0002055206250000096
defining the constructed fermentation kinetic model (formula 22-24) as a state equation (formula 25-27) of Pontrieau maximum principle, constructing an objective function (formula 28) by using the umami peptide synthesis kinetic model, and solving the corresponding fermentation control condition mu when the yield of the umami peptide is maximumsetWithin 0 to 0.6h-1Process variation (μ) within the rangeset(t)). On the basis, a Hamiltonian (expression 29) is constructed by introducing Lagrange multipliers (lambda (t)), and an adjoint (expression 30) and a control equation (expression 31) are further determined according to the Pontrieau maximum principle. Simultaneously solving the state equation, the fitness function and the control equation to determine the corresponding mu when the target function obtains the extreme valueset(t) of (d). Final muset(t) taking one point every 2h for actual control, wherein the specific change rule is shown in Table 4.
The state equation is as follows:
Figure BDA0002055206250000101
Figure BDA0002055206250000102
Figure BDA0002055206250000103
an objective function:
Figure BDA0002055206250000104
hamiltonian:
Figure BDA0002055206250000105
the adjoint function:
Figure BDA0002055206250000111
the control equation:
Figure BDA0002055206250000112
TABLE 4 mu at different stages optimized based on Pontrieau extreme value principlesetValue of
Figure BDA0002055206250000113
Example three: fermentation optimization production of delicious peptide BMP based on Pontrieau maximum principle
(1) Culture medium
The seed culture medium used was LB medium: 10g/L of peptone, 5g/L of yeast extract and 10g/L of sodium chloride. Fermentation medium (g/L): 7.5 parts of diammonium hydrogen citrate, 2.0 parts of sodium sulfate, 20.0 parts of ammonium sulfate, 3.5 parts of ammonium chloride, 14.6 parts of dipotassium phosphate, 4.0 parts of sodium dihydrogen phosphate monohydrate, 1.0 part of magnesium sulfate heptahydrate, 3.0mL parts of trace metal ion solution and 20.0 parts of glucose (separately sterilized). Trace metal ion solution (g/L): 0.5 part of calcium chloride, 0.18 part of zinc sulfate heptahydrate, 0.1 part of manganese sulfate monohydrate, 10.05 parts of ethylene diamine tetraacetic acid, 8.35 parts of ferric chloride, 0.16 part of copper sulfate pentahydrate and 0.18 part of cobalt chloride hexahydrate. Feed medium (g/L): glucose 800.
(2) Conditions of fermentation
The engineering bacteria of subtilis 168/pMA09srfA-8BMP are used as the self-induced expression bacteria of BMP and are fermented and produced in a 5L fermentation tank containing 2.5L of initial culture medium. Before the fermentation in a tank, firstly, inoculating the expression bacteria preserved in the glycerin pipe into 5mL of seed culture medium according to the proportion of 0.1%, and culturing for 8h under the conditions of 200rpm and 37 ℃ to activate the bacteria; then transferring the culture medium into a 250mL triangular flask containing 50mL seed culture medium in a proportion of 1%, and culturing for 8h under the same culture conditions; finally, the culture solution is used as a fermentation seed culture solution and is inoculated into a fermentation tank according to the proportion of 10 percent for fermentation culture. During the whole fermentation process, the culture temperature is 37 ℃, the culture pH is 7, and the DO in the fermentation liquor is maintained at about 30% by automatically adjusting the air flow rate and the stirring speed.
Optimum mu at the growth stage of the biomass index as shown in Table 4 according to the above fermentation conditionssetAnd (3) value control, namely performing multi-stage index feeding control on the fermentation of the delicious peptide BMP by combining an index fed-batch formula (formula 1). During the feeding, the exponential feeding was simulated by adjusting the feeding rate F-value once per hour. The Dry Cell Weight (DCW), glucose concentration and product concentration in the fermentor were measured every 2h to monitor the real-time fermentation (FIG. 2). After fermentation for 40h, under the control of fermentation based on the Pontrieau maximum principle, the accumulation of substrate glucose is not obvious, and the concentrations of the bacterial DCW and the product reach 71.32g/L and 3.16g/L respectively.
Measurement of the Dry weight of the cells: 5mL of the fermentation broth was taken out of the fermenter every 2h for dry weight measurement, and the fermentation broth was centrifuged in a centrifuge at 12000rpm for 20 min. Removing supernatant, re-suspending with distilled water, centrifuging again under the same condition for 20min, and drying at 80 deg.C to constant weight; the glucose concentration is automatically detected by a glucose analyzer; the product concentration was determined by the Bradford method.
Comparative example one: batch fermentation production of umami peptide BMP
Compared with the culture medium used in the batch fermentation experiment in the example (3), the concentrations of the other components are consistent with the concentration of the glucose except that the concentration of the glucose is increased to 40g/L, the fermentation conditions are also carried out according to the conditions described in the example (3), and the whole fermentation process is not subjected to any feeding control, so that the culture medium is used as a comparative experiment for optimizing the fermentation based on the Pontrieau extreme value principle. After batch fermentation for 40h, the bacterial concentration and the yield of the umami peptide were found to reach 22.53g/L and 1.11g/L, respectively (FIG. 3).
Comparing the results of fermentation of umami peptide based on the Pontrieau maximum principle with the results of batch fermentation in comparative example 1, the cell concentration and the yield of umami peptide in the former were found to be 3.2 times and 2.8 times, respectively, of the latter. The result shows that the optimized fermentation feeding control based on the Pontrieau maximum value principle is more suitable for the actual growth condition of the thallus B.subtilis 168/pMA09srfA-8BMP, can fully meet the carbon source requirement of the thallus, effectively avoids the substrate inhibition caused by carbon source accumulation, improves the yield of a target product, and lays a theoretical foundation for the large-scale production of the BMP and other umami peptides or proteins.
While the present invention has been described in detail with reference to the embodiments, it should not be construed as limited to the scope of the patent. Various modifications and changes may be made by those skilled in the art without inventive step within the scope of the appended claims.
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Claims (2)

1. A method for producing umami peptide by fermentation based on the principle of exponential flow feeding and supplementing materials is characterized in that: inoculating 10-15% of biological expression engineering bacteria of umami peptide into a culture medium with pH 7 in a volume ratio, and fermenting at 37 ℃, wherein the umami peptide is beef flavor peptide, the sequence of the umami peptide is shown in SEQ ID NO.1, the expression engineering bacteria is B.subtilis 168/pMA09srfA-8BMP, and the culture medium comprises the following components in concentration: 7.5g/L diammonium hydrogen citrate, 2.0g/L sodium sulfate, 20.0g/L ammonium sulfate, 3.5g/L ammonium chloride, 14.6g/L dipotassium phosphate, 4.0g/L sodium dihydrogen phosphate monohydrate, 1.0g/L magnesium sulfate heptahydrate, 20.0g/L glucose and 3.0mL/L trace metal ion solution; the feeding flow rate in the fermentation process is determined by the formula 1,
Figure FDA0002750872010000011
wherein F represents the feed addition rate, μsetShowing the specific growth rate of the cells, X0Shows the cell concentration in the fermenter at the start of replenishing the feed, V0Denotes the volume of medium in the fermenter at the start of the feed, SFDenotes the substrate glucose concentration in the feed medium, YX/SThe yield coefficient of the thallus growth based on a substrate, and t represents the feeding time; the specific growth rate of the thalli is musetOptimized by Pontrieau maximum principle to obtain the specific growth rate mu of the thallisetThe optimization comprises the following steps:
s1: establishing a fermentation kinetic model of the umami peptide biological expression engineering bacteria, wherein the model is shown as a formula 2-4;
Figure FDA0002750872010000012
Figure FDA0002750872010000013
Figure FDA0002750872010000021
wherein, the formula 2 is a thallus growth rate formulaEquation 3 is a substrate consumption rate equation, and equation 4 is a product synthesis rate equation; x is the thallus density in the fermenter during the feed-batch fermentation, S is the glucose concentration in the fermenter during the feed-batch fermentation, P is the product concentration in the fermenter during the feed-batch fermentation, YP/SFor the product substrate-based yield coefficient, qmFor a specific substrate consumption rate coefficient for cell maintenance, α is a product expression rate coefficient related to cell growth rate and β is a cell density-related product expression rate coefficient;
s2: determining a state equation, an objective function and a control condition to be solved in the Pontrieau maximum principle based on the fermentation kinetic model of S1;
Figure FDA0002750872010000022
Figure FDA0002750872010000023
Figure FDA0002750872010000024
Figure FDA0002750872010000025
wherein, the equations 5-7 are state equations, and the equation 8 is a target function;
s3: based on the state equation and the objective function in step S2, introducing a lagrange multiplier to construct a hamiltonian, and determining necessary conditions for extremum calculation of the objective function: an adjoint function and a governing equation;
H＝λ1f1+λ2f2+(1+λ3)f3 9
Figure FDA0002750872010000031
Figure FDA0002750872010000032
wherein, formula 9 is a Hamiltonian, formula 10 is an adjoint function, and formula 11 is a control equation;
s4: simultaneously solving the state equation, the adjoint function and the control equation in the steps S2 and S3, determining the optimal process change of the corresponding control condition when the objective function obtains an extreme value in a given condition range, namely, optimizing to obtain the specific growth rate mu of different time periodsset。
2. The method for the fermentative production of umami peptide based on the exponential flow feed supplement principle according to claim 1, wherein the method comprises the following steps: the trace metal ion solution comprises the following components in concentration: 0.5g/L of calcium chloride, 0.18g/L of zinc sulfate heptahydrate, 0.1g/L of manganese sulfate monohydrate, 10.05g/L of disodium ethylene diamine tetraacetate, 8.35g/L of ferric chloride, 0.16g/L of copper sulfate pentahydrate and 0.18g/L of cobalt chloride hexahydrate.
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