Enzymes and its corresponding maximum conversion of substrates based on previous studies.
\r\n\t
",isbn:"978-1-83881-920-0",printIsbn:"978-1-83881-919-4",pdfIsbn:"978-1-83881-933-0",doi:null,price:0,priceEur:0,priceUsd:0,slug:null,numberOfPages:0,isOpenForSubmission:!1,hash:"c1999f94cbb4a0340032feb750236ffa",bookSignature:"Prof. Zamzuri Idris",publishedDate:null,coverURL:"https://cdn.intechopen.com/books/images_new/9656.jpg",keywords:"Paediatric Brain Injury, Adult Brain Injury, Brain Swelling, Immune Response, Brainwaves, Endocrine, Neuromonitoring, Intensive Care, Hypothermia, Neuroimaging, Brain-computer Interface, Robotic Rehabilitation",numberOfDownloads:1131,numberOfWosCitations:0,numberOfCrossrefCitations:0,numberOfDimensionsCitations:0,numberOfTotalCitations:0,isAvailableForWebshopOrdering:!0,dateEndFirstStepPublish:"June 2nd 2020",dateEndSecondStepPublish:"September 9th 2020",dateEndThirdStepPublish:"November 8th 2020",dateEndFourthStepPublish:"January 27th 2021",dateEndFifthStepPublish:"March 28th 2021",remainingDaysToSecondStep:"7 months",secondStepPassed:!0,currentStepOfPublishingProcess:5,editedByType:null,kuFlag:!1,biosketch:"Dr. Idris is acting as the Head of the Committee in Malaysia Neurosurgical Teaching and Education, Head for the Local Neurosurgical Association of Malaysia, Member for the Society for Brain Mapping and Therapeutics USA, for the European Society for Stereotactic and Functional Neurosurgery. He has graduated from the University of Wales College of Medicine Cardiff, then pursued his postgraduate career at Universiti Sains Malaysia. He completed his fellowship program at the University of Gent, Belgium.",coeditorOneBiosketch:null,coeditorTwoBiosketch:null,coeditorThreeBiosketch:null,coeditorFourBiosketch:null,coeditorFiveBiosketch:null,editors:[{id:"73844",title:"Prof.",name:"Zamzuri",middleName:null,surname:"Idris",slug:"zamzuri-idris",fullName:"Zamzuri Idris",profilePictureURL:"https://mts.intechopen.com/storage/users/73844/images/system/73844.png",biography:"Prof Dr Zamzuri Idris had graduated from University of Wales College of Medicine Cardiff UK in 1994, then pursued his postgraduate career in Neurosurgery locally at Universiti Sains Malaysia. In 2007, he completed his fellowship programme in Gent Belgium under Prof J. Caemaert in Neuroendoscopy and Prof D. Van Roost in Functional Neurosurgery. He is currently the Head of Neuroscience Department and holds various important posts related to Neurosurgery, Neurosciences and Scientific Society. He is currently acting as the Head of the Committee in Malaysia Neurosurgical Teaching and Education, Head for the Local Neurosurgical-Board Examination and an Executive Committee Member for Neurosurgical Association of Malaysia, Member for the Society for Brain Mapping and Therapeutics USA, for the European Society for Stereotactic and Functional Neurosurgery (ESSFN) and for the Asia Pacific Low Grade Glioma Network. His major interests are in brain trauma, minimally invasive neurosurgery (neuroendoscopy), precision radiation therapy, epilepsy, brain mapping, brain stimulation, brainwaves and lately in Quantum Brain. 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Many simple esters are pleasant-smelling liquids and mainly used as fragrant odors of fruits and flowers. For example, methyl butanoate is an element found in pineapple oil, whereas isoamyl acetate is an element of banana oil [1]. These esters are also naturally present in animal fats and oil [2] and in many biologically important molecules. Esters are ubiquitous and contain “nature-identical” substance that can be used to substitute natural flavor and fragrances. The demand for flavor and fragrance products is fairly high for most applications in developed countries. In 2009, flavor and fragrance industry faced a decline due to global economic crisis, but rapidly recovered a year after. The market was forecasted to continue expanding at a CAGR of 5.6% during 2011–2013 [3].
As the demand on flavored food increased tremendously throughout the years, consumers were also concerned about the natural ingredients of it by considering food with natural flavored in their list. The term “natural” has been clearly defined by the U.S. Code of Federal Regulations 101.22(a)(3) as “…the essential oil, oleoresin, essence or extractive, protein hydrolysate (product of hydrolysis), distillate of any product of roasting, heating or enzymolysis, which contains the flavoring constituents derived from a spice, fruit juice, vegetable or vegetable juice, edible yeast, herb, bud, bark, root, leaf or similar plant material, meat, seafood, poultry, eggs, dairy products or fermentation products thereof, whose significant function in food is imparting flavoring rather than nutritional” [4].
Esters naturally available in plants and flowers were extracted for traditional flavor production. However, the traditional extraction of flavor from plants is too expensive for commercial exploitation, limitation of raw materials, and only small amount of esters produced. On the other hand, the demand of esters kept increasing; therefore, researchers overcome the problems with alternative production route via chemical synthesis. Esterification via chemical synthesis is based on Fischer esterification method. Its drawbacks attributed to the chemicals used and consumers’ awareness toward chemicals added to their food makes the synthesis is not favored in the food industry. Hence, a new method of ester synthesis is required to produce large number of esters for industrial application with high economic benefit and a purer end product.
Synthesis of isoamyl acetate in organic solvent has been introduced. Due to region- and stereo-specificity expressed by most lipases in mild operation conditions and high degree of purity produced, lipase-catalyzed esterification in organic solvent has recently received greater consideration relative to the traditional chemical synthetic methods, particularly in the production of natural flavor and fragrance. Despite the higher conversion yields of esters, organic solvents undoubtedly bring about negative impact on solvent toxicity, inflammable, and need extra action on separation process. In addition, some organic solvents used are too expensive to allow profitable commercial scale-up [5]. Hence, a solvent-free system was introduced in the esterification process.
The absence of solvents in the solvent-free synthesis gives advantages on the downstream processing as there would be fewer components present in the reaction mixture at the end of the esterification process. Moreover, the production cost can be minimized. In addition, Yahya et al. [6] has stated that it is possible to use high substrates’ concentrations in a solvent-free system. Hence, it is scientifically and environmentally wise to produce ester via solvent-free biotechnological route that would eliminate all the disadvantages of traditional and chemical synthesis route of producing esters.
Enzymes react by accelerating both the rate and specificity of metabolic reactions of the substrates without changing its original shape and amount [5]. In the food industry, flavor production from enzyme-catalyzed process has been reviewed by Christen and Munguia [7]. Common types of enzymes used in food flavor are hydrolases and oxidoreductases [7, 8]. One of the hydrolases enzymes is lipase. Lipase is also capable to form ester bounds under reverse hydrolytic conditions which allow catalysis of various other types of esters [9]. The demand for natural and environmental friendly product is the main key point of the usage of lipase in ester synthesis. Therefore, lipases are considered as enzymes of high commercial potential due to their flexibility in application. There are several lipases which have been used in isoamyl acetate production. Summary on the isoamyl acetate production based on different enzymes is shown in Table 1.
Lipase origins | Maximum conversion, % | References |
---|---|---|
95.5 | Gubicza et al. [10] | |
80 | Guvenc et al. [11] | |
100 | Romero et al. [12] | |
96 | Romero et al. [13] | |
100 | Feher et al. [14] | |
∼99 | Wolfson et al. [15] | |
>90 | Krishna et al. [16] | |
∼40 | Romero et al. [13] | |
>80 | Krishna et al. [17] | |
96.4 | Mittelbach and Trathnigg [18] | |
64 | Ghamgui et al. [19] | |
∼42 | Romero et al. [13] |
Enzymes and its corresponding maximum conversion of substrates based on previous studies.
From Table 1, it can be observed that a lot of works have been done by using
Recyclability of enzymes is also an important factor to be considered for industrial-scale applications as it can reduce the cost of raw materials. For esterification in the organic solvent,
In this study, isoamyl acetate was produced experimentally by reacting acetic anhydride and isoamyl alcohol with the presence of enzyme,
Materials/chemicals | Purity | Usage | Supplier |
---|---|---|---|
Isoamyl alcohol | 99.8% | Production medium | Merck Co., Malaysia |
Acetic anhydride | 98% | Production medium | ACROS Organics, Malaysia |
Isoamyl acetate | 100% | GC standard | Merck Co., Malaysia |
CALB (≥5000 U/g) | N/A | Production medium | Sigma-Aldrich, Malaysia |
List of materials and chemicals used.
The production of isoamyl acetate enzymatic synthesis from acetic anhydride was done in lab scale. All of the experimental works were carried out using 100-ml Erlenmeyer flasks with stopped rubber, which then were placed in an incubator shaker (Benchmark Incu-shaker mini, New Jersey). Incubator shaker was used to maintain the mixing rate and to control the temperature. Then, flame ionization detector gas chromatography (GC-FID) (Agilent Technologies, 7820A GC system, USA) was used to analyze the concentrations of compounds in the sample taken.
Isoamyl acetate syntheses were carried out without any organic solvent in 100-ml stopped-rubber Erlenmeyer flask with working volume of 15 ml. Enzyme was added into the reaction media containing a mixture of isoamyl alcohol and acetic anhydride at various temperatures. The reaction mixture was then incubated in an incubator shaker (Benchmark) at 150 rpm for 6 h. The basis of this experimental method was taken from [16].
About 0.5 ml of the reaction mixture was withdrawn periodically starting from t = 0 h, until t = 6 h for analysis. The withdrawal was done using micropipette and transferred into microcentrifuge tube. Samples were analyzed using gas chromatograph (Agilent Technologies 7820A) equipped with a hydrogen flame ionization detector and a SGE BP21 (FFAP) column (60 m × 0.32 mm × 0.25 μm). Helium was used as a carrier gas at a flow rate of 5 ml/min. After injection of samples, the oven temperature was kept at 100°C and linearly increased to 140°C. The rate of temperature increase was set at 70°C/min, and was kept at 140°C for the remaining time of analysis. Injector and detector temperatures were set at 200 and 250°C, respectively.
Quantification of data was done by calibration with standards samples. Each sample required 4.08 min to be analyzed by GC-FID. The retention times of peaks were as follows: isoamyl acetate, 2.26 min; isoamyl alcohol, 2.38 min; acetic anhydride, 2.48 min; and acetic acid, 3.2 min.
The effects of reaction temperature on the enzymatic esterification were studied at various temperatures: 30, 40, and 50°C. About 15 ml working volume of the medium in a 100-ml Erlenmeyer flask was incubated in an incubator shaker with agitation speed of 150 rpm for 6 h reaction time. Samples were taken periodically until 6 h of reaction time and analyzed by GC-FID for isoamyl acetate production.
The effect of acid/alcohol molar ratio was studied at various acid/alcohol molar ratios: 0.1 (excess alcohol), 1 (equimolar), and 2 (excess acid). The medium was incubated in an incubator shaker at 40°C reaction temperature, and with agitation speed of 150 rpm for 6 h reaction time. Samples were taken periodically until 6 h of reaction time and analyzed with GC-FID for isoamyl acetate content.
The enzyme loading effects were studied at various percentages of enzymes in medium: 4, 8, and 12%. The calculation was based on the overall mass of substrates used in the reaction. The medium together with the enzyme was incubated in an incubator shaker at a temperature of 40°C. The agitation speed was set to 150 rpm; samples were taken at different time intervals until 6 h of reaction time and were analyzed using GC-FID for isoamyl acetate content.
Optimization studies are carried out using response surface methodology (RSM). RSM is a collection of statistical and mathematical analysis for developing, improving, and optimizing processes in which the response developed is influenced by several variables. It has an important application in the process development, design, and formulation of new products, as well as in the improvement of existing product design.
Optimum conditions for isoamyl acetate enzymatic esterification can be obtained by using optimization software, Design Expert 6.0.6. The method used was Central Composite Design (CCD), under RSM. CCD is the best design for response optimization [21]. In this study, three levels and four factor variables were chosen. The three levels represent the three points between the lower and upper limit of the parameters, whereas the four factors represent the four parameters that are studied in this section, which are reaction temperature, ac/al molar ratio, enzyme loading, and reaction time. The details of the parameters and levels studied were shown in Table 3.
Levels | |||||
---|---|---|---|---|---|
Variables | Coding | Unit | −1 | 0 | +1 |
Temperature | A | °C | 30.00 | 40.00 | 50.00 |
Ac/Al ratio | B | — | 0.1 | 1 | 2 |
Enzyme loading | C | % | 4 | 8 | 12 |
Reaction time | D | h | 2 | 4 | 6 |
List of variables and its value.
Sensitivity analysis is useful for testing the robustness of the result of a model developed, to show the relationships between input and output variables in a system, and for model simplification by removing the insensitive or insignificant variables.
Based on the optimization step before, a sensitivity analysis for each individual parameter and the interaction between parameters in this study were done using application tools provided by RSM. It was done to decide the interaction between parameters, and the most sensitive parameters in esterification process.
RSM model was developed using DoE software by designing new experiments for enzyme kinetic model. Three-level and four-factor designs which consist of 27 sets of experiment were constructed using parameters listed in Table 4.
Variables | Coding | Unit | Levels | ||
---|---|---|---|---|---|
−1 | 0 | +1 | |||
Temperature | °C | 30 | 40 | 50 | |
Mass enzyme | wt% | 4 | 8 | 12 | |
Reaction time | h | 2 | 4 | 6 | |
Reciprocal of anhydride concentration | l/mol | 0.12 | 0.20 | 1.18 |
Experimental range and levels of variables.
First principle model is an application of conservation of mass to the analysis of a physical system by taking account of material entering, leaving, generating, consuming, and accumulating in the system.
The mathematical general equation of a balanced mass conservation quantity by using conservation law in a system is:
In term of general mole balance, the above equation became
By assuming component
In a close system (batch process), assuming with well stirred substrate, the above equation reduces to:
Therefore,
where
The reaction rates can be derived in detail by enzyme kinetic equation.
The entire model developed will then need to be validated to assure the models are reliable and can be used in industrial application. Validation of the kinetic model was done by comparing the output from the model developed with the experimental data collected. The results were compared and plotted in a graph, and error value based on the regression analysis was done.
Theoretically, acetic anhydride possesses two acyl groups. In a reaction with isoamyl alcohol, one of the acyl from acetic anhydride will bind with isoamyl alcohol and discharge one H+ to form isoamyl acetate and acetic acid. Then, excess acyl (from acetic acid) will react with excess alcohol to form another isoamyl acetate and water. The details of reaction scheme are shown below:
First reaction
Second reaction
Overall reaction
Based on the chemical reaction scheme shown previously, esterification of isoamyl acetate undergo two reactions, first is between the acetic anhydride and isoamyl alcohol, producing acetic acid and isoamyl acetate; and the second reaction is between the acetic acid and excess isoamyl alcohol, producing another isoamyl acetate and water as by-product. An overall analysis on the effect of substrates concentration was done at 8% enzyme loading, 6 h of reaction time at acid/alcohol ratio of 0.1, 1, and 2, and the results were plotted in Figures 1, 2, 3 respectively.
Overall concentration of substrates and product during the reaction for acid/alcohol ratio of 0.1.
Overall concentration of substrates and product during the reaction for acid/alcohol ratio of 1.0.
Overall concentration of substrates and product during the reaction for acid/alcohol ratio of 2.0.
From Figure 1, the concentration of isoamyl acetate increased rapidly at the initial of the reaction until 15 min of reaction time. During that time, acetic anhydride was consumed until 98% of its initial concentration, whereas the concentration of acetic acid and isoamyl acetate was increasing. This is in line with the reaction mechanism involved, where the reaction between acetic anhydride and isoamyl alcohol will produce acetic acid and isoamyl acetate initially. As clearly shown in Figure 1, as acetic anhydride was completely consumed, acetic acid produced will then react with the excess isoamyl alcohol, producing isoamyl acetate and water. This is evident by the reducing of acetic acid concentration after 15 min of reaction time and consequently the rapid increase of isoamyl acetate concentration. Throughout that time, concentration of acetic acid starts decreasing until 80% of acetic acid’s initial production. At the end of the 6-h reaction time, isoamyl alcohol was in excess, acetic anhydride and acetic acid were 98 and 80% consumed, respectively.
From Figure 2 initially, concentration of isoamyl acetate increased rapidly at the first 30 min of reaction time. During that time, acetic anhydride was consumed until 95% of its initial concentration, whereas concentration of acetic acid and isoamyl acetate was increasing, following the reaction mechanism described by the above reaction equation, which is reaction between acetic anhydride and isoamyl alcohol will initially produce acetic acid and isoamyl acetate. Acetic anhydride was fully consumed during 180 min of reaction time, and acetic acid produced at the beginning of reaction was in excess until the end of 6 h reaction duration as well as isoamyl alcohol. This is because the production of isoamyl acetate has achieved steady state, and the excess amount of alcohol is lower than the minimum amount which required to be reacted.
Figure 3 clearly shows that in excess acetic anhydride condition, isoamyl alcohol was 98% consumed during the 240 min of reaction time. After that, there is no decreasing trend in either acetic anhydride or acetic acid concentration. Acetic acid produced by the primary reaction at the beginning of the test was in excess since isoamyl alcohol is in limited condition. This shows that both reactions were stopped by limited amount of isoamyl alcohol in the mixture.
Based on Figures 1, 2, 3, isoamyl alcohol is the critical substrate in this esterification reaction. The final amount of isoamyl acetate produced is highly depending on the amount of isoamyl alcohol at the initial of the reaction. Limited amount of isoamyl alcohol will automatically stop the reaction mechanism, since there is no receiver of acyl in the mixtures. This also shows that acetic anhydride amount in the mixtures need to be restricted to some amount so that there is no excess acyl in the reaction, and hence making this process cost-effective [22].
Reaction temperature of esterification process has an effect on the final yield of isoamyl acetate produced based on the Arrhenius equation (Eq. (6)).
where
Eq. (6) clearly shows that reaction temperature has parallel effect to the reaction rate constant and hence influences the final yield of isoamyl acetate produced by affecting the esterification reaction rate.
Effect of temperature on the yield of ester produced has been investigated at temperatures 30, 40, and 50°C for 6 h of reaction time. From literatures, the range of temperature studied was between 30 and 65°C. However, the optimum reaction temperature was found to be between 30 and 50°C. Hence, to elucidate the impact of reaction temperature on yield of ester, the synthesis has been studied at a temperature range from 30 to 50°C for 6 h of reaction time, at 8% enzyme concentration and acid/alcohol molar ratio of 1. The results are illustrated in Figure 4.
Effect of temperature on the ester production.
Based on Figure 4, initial reaction rate of ester production increased with increasing reaction temperature from 30 to 50°C. This would be explained by Eq. (6), where increasing reaction temperature would increase the kinetics of the reaction, hence encourage the collision rate between molecules in the medium, and thus favor higher production of ester. This result is in agreement with a research done by [23, 24], where increasing reaction temperature will increase the reaction rate and hence produce higher concentration of ester. Similar result on the positive effect of reaction kinetics toward the increasing of reaction temperature was also found by the studies.
As the reaction time increased, the production rate of ester appears to decrease for reaction temperatures of 40 and 50°C compared to the production at 30°C. The final yield of ester produced at 30, 40, and 50°C of reaction temperatures at time 6 h of reaction time were 67.2, 61.8, and 59.1%, respectively. This could be due to the enzyme tertiary structure that starts to disrupt at higher reaction temperature and at longer reaction time, hence losing its catalytic activity, thus lowering the enzyme production rate [25].
The effect of acid/alcohol molar ratio has been studied at low anhydride concentration, equimolar, and excess in anhydride, whose ratios were 0.1, 1, and 2, respectively. The reaction temperature was set at 40°C and enzyme loading at 8%. The result for this experimental condition is shown in Figure 5.
Effect of acid-alcohol ratio on the yield of ester.
Based on Figure 5, a maximum yield of isoamyl acetate was achieved when isoamyl alcohol is in excess over the acetic anhydride ratio. This is due to the availability of excess nucleophile in the reaction mixture. Based on the reaction scheme shown in Section 4.1, an acyl from acetic anhydride is reacted with a nucleophile available from isoamyl alcohol and producing isoamyl acetate and acetic acid. Then, an acyl from acetic acid produced reacted for the second time with the available nucleophile in the reaction mixture and produced another isoamyl acetate and water. The maximum yield of isoamyl acetate produced at acid/alcohol molar ratios of 0.1, 1, and 2 was 128, 53, and 88%, respectively.
The current study is in agreement with [14] and [26], where higher substrate concentration (high acid/alcohol molar ratio) leads to lower yields of ester. This could also be due to acid concentration which has met the critical concentration needed in the reaction [27]. However, by increasing the molar ratio to excess acid, the yield of ester starts to increase again. This condition happened because the concentration of acid in the reaction mixture has past the critical concentration of acid needed in the reaction medium.
The effect of enzyme loading concentration was studied at 4, 8, and 12% of enzyme loading, acid/alcohol molar ratio of 1 and 30°C of reaction temperature. Enzyme loading is economically important to the esterification process as enzyme is costly compared to other materials used in the synthesis. Producing high yield of isoamyl acetate at low quantity of enzyme synthesis was highly preferred in an esterification synthesis. Therefore, in this study, the yield of isoamyl acetate produced versus reaction time was plotted and shown in Figure 6 for three different enzyme loadings.
Effect of enzyme loading on the ester production.
Based on the figure above, increasing amount of enzyme loading increases the yield of ester produced. The maximum yield of isoamyl acetate produced by the synthesis for 4, 8, and 12% are 57, 65.8, and 71.5%, respectively. The graph also shows the clearer trend of enzyme loading effect along the 6 h of reaction time. For lower amount of enzyme loading, longer reaction needed to achieve steady state of the esterification reaction. Increasing the enzyme loading from 4 to 12% shortened the time required for the reaction to achieve steady state which is favorable. It also shows that esterification reaction increases with the increasing amount of enzyme loading and the trends resembles the results of esterification studied by [26]. This concludes that the rate of esterification is dependent on the enzyme concentration used in the mixture.
Design Expert software contained many types of optimization methods that can be used in this study. Based on the optimization methods described, a central composite design (CCD) method was chosen to design all the experiments conducted for esterification synthesis. CCD is generally the best design for response optimization, as stated by [21, 28]. In this study, three-level and four-factor designs were used to determine the optimum condition with four experimental parameters (temperature, acid-alcohol molar ratio, enzyme ratio, and reaction time) and three-level indicated the level of each range (−1, 0, +1). Six replicates which run at the center point (0, 0) of the design were performed to allow the estimation of pure error. All the experiments were carried out in the randomized order to minimize the unexplained variability in the observed responses due to irrelevant factor.
Fitting of the data to models in RSM and their subsequent ANOVA showed by Figure 7 confirmed that the enzymatic esterification reaction of acetic anhydride and isoamyl alcohol was most suitably described by quadratic model. The equation of the model based on the actual values is shown by Eq. (7).
Parity plot for relation between observed and predicted isoamyl acetate synthesis.
The quadratic response function represents the yield of ester produced, where
Closer the value of
Statistical analysis from the analysis of variance (ANOVA) was done using RSM software (result not shown). The
Model developed by RSM needs to be validated to make sure it is reliable and acceptable. Figure 8 shows the comparison of ester yield from the actual experiment and calculated by model developed. The comparison was done for 27 runs of experiments.
Comparison between the RSM model developed and the actual experimental data.
Based on Figure 8, the yield of ester’s model developed by RSM appear to fit well with the actual results of ester yield from the experimental data. This clearly shows that the model developed is reliable to use as prediction of the real process of enzymatic esterification synthesis between acetic anhydride and isoamyl alcohol catalyzed by CALB in solvent-free system.
Numerical optimization was done by setting the desired optimum condition and product, and then DoE software will generate few solutions based on the model developed before. Based on the desired optimized condition setting, four sets of solutions for optimized experimental condition for enzymatic esterification in SFS were developed as shown in Table 5.
Parameters | Constrains | Solution | ||
---|---|---|---|---|
Goal | Lower limit | Upper limit | ||
T (°C) | Minimize | 30 | 50 | 30 |
Ac/Al | Is in range | 0.1 | 2 | 0.10 |
Enzyme (%) | Minimize | 4 | 12 | 4.14 |
Time (h) | Minimize | 2 | 6 | 2.00 |
Yield (%) | Maximize | 52.00 | 130.21 | 112.83 |
Optimized condition suggested by DoE software.
Based on the numerical optimization, the most desirable reaction conditions for optimum isoamyl acetate yield was set at minimum reaction temperature, enzyme loading, and reaction time. Minimum reaction temperature is required to reduce the energy usage, hence lower the operating cost of esterification process. Enzyme loading was set to minimum in order to reduce the usage of enzyme in esterification synthesis as enzyme is the most expensive material in esterification synthesis. Lowering reaction time to the minimum can help to accelerate the production rate of isoamyl acetate and give benefit to the industry. The results for optimum condition are shown in Table 6.
Number | Temperature (°C) | Ac/Al ratio | Enzyme loading (%) | Reaction time (h) | Yield (%) | Error ( | |
---|---|---|---|---|---|---|---|
Predicted | Actual | ||||||
1 | 30.0 | 0.10 | 4.14 | 2.0 | 112.83 | 112.63 | 0.18 |
Result and error analysis of model validation.
Experimental result showed that there was no significant difference for percentage yield of ester produces between the predicted and actual values. The error value for the optimization experiment is less than 1%. Therefore, the model obtained is reliable to predict the yield of isoamyl acetate in enzymatic esterification of isoamyl acetate by solvent-free system with high accuracy.
Design of experiment (DoE) software is used again to design the experiment. Three-level and four-factor designs which consist of 27 sets of experiments were done. The effect of temperature,
All coefficients obtained from the full quadratic polynomial model were evaluated by regression analysis and tested for their significance. The insignificant coefficients were eliminated based on
The final model for reciprocal of enzymatic reaction rate obtained from the CCD analysis is:
By using Eq. (8), the general equation for reaction rate developed throughout the experiment is given by:
Given that general reaction rate equation,
where
Since the overall reaction is reversible, hence Eq. (10) becomes
where
Overall reaction for isoamyl acetate esterification from acetic anhydride and isoamyl alcohol is:
Based on Eq. (11), there were few reaction rate equations that are possible to be applied for this isoamyl acetate enzymatic esterification reaction; therefore, all of the possible reaction rate equations were listed in Table 7 and reaction rate constants for all equation developed were solved out using nonlinear equation solver in POLYMATH and the regression of each equation was compared. Table 8 shows that the accurate reaction rate equation was for the third reaction rate with
No. | Reaction rate equation | ||||
---|---|---|---|---|---|
1 | 0.0461 | 0.3984 | 0.5705 | 0.5533 | |
2 | 0.0078 | 0.2426 | 0.9121 | 0.9086 | |
3 | −0.0135 | 0.2530 | 0.9385 | 0.9360 | |
4 | 0.0013 | 0.2447 | 0.9109 | 0.9073 | |
5 | 0.0187 | 0.0912 | 0.8111 | 0.8035 | |
6 | 0.0032 | 0.0961 | 0.7864 | 0.7778 | |
7 | 0.0172 | 0.0951 | 0.7890 | 0.7806 | |
8 | 0.1912 | 0.0909 | 0.7936 | 0.7853 | |
9 | −0.0147 | 0.2474 | 0.9109 | 0.9073 |
Possible reaction rate equation, constant developed, and regression analysis.
Model | R2 |
---|---|
RSM model | 0.90 |
First principle model | 0.89 |
Regression analysis between the model developed and the actual data from experimental results.
where
Validation of kinetic equation was done by comparing the experimental data for reaction rate of each run with the reaction rate calculated by Eqs. (8) and (12). Then, the results were plotted in Figure 9 and regression analysis was done to evaluate the accuracy of models developed.
Comparison between the actual reaction rate and the modeled reaction rate.
Regression analysis was done between the models and the actual data by using excel and the result of regression analysis is shown in Table 8. The regression value (
In this study, it was concluded that there are two chemical reactions involved in the esterification of isoamyl acetate from acetic anhydride and isoamyl alcohol. The main reaction is between acetic anhydride and isoamyl alcohol, and the combination of the two reactions results in an overall reaction as follow:
Between all of the parameters studied, the most critical parameter in isoamyl acetate synthesis is the acid/alcohol molar ratio. This is because the molar ratio will affect the nucleophile and acyl content in the mixture. The least sensitive parameter is the reaction temperature of the ester synthesis. This can be shown by small gap on the yield of ester produced at large different of reaction temperature.
Optimization of enzymatic isoamyl acetate synthesis in solvent-free system was done using RSM. The present process fits well with second order quadratic equation with determination of coefficient (
Author would like to acknowledge Ministry of Education Malaysia for the financial support through MyBrain15 scheme.
The warm-up is widely understood as a preparation practice to perform before any physical exercise. It is usually used by athletes, coaches, and general physical activity participants, to obtain an optimal physical and psychological state and to get kinetic and coordinative preparation in the prevention of injuries during the practice [1, 2, 3, 4].
Based on previous studies, the main benefits of the warm-up were increased body temperature, decreased muscle and joint stiffness, [5] increased efficiency in the transmission of nerve impulses, [6] and, simultaneously, the increase in metabolic reactions, leading to the improvement of muscle power. [7] It may also lead to an increase in the dissociation of oxygen, hemoglobin, and myoglobin, causing vasodilation and, consequently, an increase in muscle blood flow. [8] These changes could be promoted by two basic types of warm-up, specifically, the active and passive warm-up. [3, 9] Hot water bags, short waves, hot baths, sauna, are some of the means used to complete a passive warm-up. [3, 9] This type of warm-up provides an increase in muscle and central temperature without energy expenditure, with the use of external heating. [3, 9] On the other hand, the active warm-up can be performed through the use of physical activity, for instance, walking, running, swimming, cycling, or any other specific exercises. [3, 9, 10] One of the main advantages of the active warm-up is its specificity, as it prepares the muscles that will be used during the activity and could benefit from the movement itself [3, 9].
Despite the positive influence of warm-up on sports performance, [9] there is still a lack of specific investigations about the variables that compose it, the optimal warm-up design as well as its effects on the force production and strength training performance. [11, 12] Any movement performed during physical activity requires the use of specific muscles to produce movement. The movement depends on muscle performance and therefore force production, either in maximal or submaximal efforts so that the exercise could be carried out successfully. The role of muscle strength performance is widely recognized in the scientific and sports context. [9, 13] Maximizing the strength and optimizing force production should be a priority to any person participating or willing to participate in sports performance or physical exercise. For this performance improvement, in physical activity and sport context, resistance training (strength training exercises where muscles exert a force against an external load) assumes an important role to develop individual capacities. Moreover, to improve the efficiency of resistance training and force production, the warm-up could be essential. It is important to understand the way that warm-ups can influence strength training and performance, to analyze the effects, and then to provide a useful strategy to apply in the real context. With this knowledge, professionals are able to design a warm-up that will optimize resistance training and thus, maximizing strength gains, force production, and resulting in improvements in physical exercise performance.
It is then important to understand the effect of warm-up in strength performance and this may be through the assessment of maximum dynamic strength (load at 1 repetition maximum: 1RM), isometric strength, or even through the rate of production of muscle strength. [3, 10, 11, 12, 14] Previous findings suggested that the warm-up procedure (for example, aerobic exercise, specific activity, and stretching) seems to influence the results of the 1RM assessment, as well as to improve the strength produced during the assessments. [3, 12, 15] Generally, it is recommended that the warm-up routine prior to a 1RM test includes general (aerobic) and specific (imitating target activity) exercises. [16, 17, 18] The general warm-up is usually completed using an aerobic activity of low to moderate intensity with the main purpose to increase the muscle temperature, which can be performed with different types of aerobic activity (for example, running or cycling). [3, 10, 12] Stretching exercises can also be performed as part of a typical warm-up routine. Regarding the specific warm-up, it is recommended to perform it by including exercises that use the same or similar movements as the main activity at progressively higher intensities in an attempt to increase neuromuscular activation. [2, 12] In fact, there is suitable scientific evidence in the literature to support the implementation of only specific warm-ups before exercise, [19, 20] however, the effects of general warm-up on strength measurements are not clear yet.
In order to design an effective warm-up, several parameters and variables are associated with it, which seems to be extremely difficult to select an ideal type of warm-up for all sports. Then, it is necessary to understand what type of warm-up is more appropriate to the variable that influences performance in all exercises, i.e. force production. Thus, our narrative review aimed to analyze and discuss the main results of the literature on the effects of warm-up on force production and strength, by analyzing responses during resistance training and assessments of maximum strength. The results determined in this study, aimed to elucidate sport-related professionals about the effects of warm-up and help them to design their training.
The current study intended to summarize the findings and evidence reported in the literature about the effect of warm-up protocols in force production, strength evaluation and resistance training. In order to identify relevant articles on this topic, an extensive bibliographic search was carried out. Of all the articles identified, only nine were chosen, which corresponded to the theme addressed here.
A search in the literature that studied different types of warm-up was conducted, where the focus was to understand the effects of warm-up in strength performance. Considering that active warm-up is the most commonly used by people engaged in sports and physical exercise and that is the most investigated, it was only included original articles that focused on the effects of active warm-up. Original research articles published between 2010 and 2020 were selected to identify studies in which warm-up and strength performance were reported. The search for scientific articles was performed in 4 databases (Web of Science, Scopus, PubMed and ScienceDirect) in which the keywords “warming-up”, “resistance training” and “strength” with multiple combinations were used and with no restrictions of language.
To carry out this research, the studies had to respect inclusion criteria such as, being focused on active warm-up, being cross-sectional studies, focusing on measures of strength, being carried out by healthy individuals, aged 18 years or over. As exclusion criteria, all types of review (qualitative review, systematic review and meta-analysis) were excluded, the non-use of at least one active warm-up and studies with young participants (<18 years old). Articles that were not written in English were also excluded.
The literature search found 163 relevant articles, of which 152 did not meet the defined inclusion criteria. These studies were excluded based on the focus on other physical activities rather than strength-related ones, such as running performance, anthropometric characteristics, or strength evaluation performed in participants of other chronological ages including children. Consequently, a total of 11 studies were considered for further analysis. These studies were published between 2009 and 2020. The studies focus on the results that different types of warm-ups may cause in resistance training (Table 1).
Authors | Objective | Sample | Warm-ups | Main outcomes |
---|---|---|---|---|
Ribeiro et al. [21] | Verify the effects of three specific warm-ups on squat and bench press resistance training. | 14 males | 3 protocols:
| The results showed that the strength outputs were optimized mainly by warm-up with 80% of the training load in the squat training and by the warm-up that brought the two loads together (40% and 80%) in the bench press training. |
Krzysztofik and Wilk [22] | Determine the effects of plyometric push-ups as a conditioning activity on high-loaded bench press performance. | 24 males | 2 protocols:
| The results demonstrated that plyometric push-ups lead to performance enhancement of the bench press exercise at 70%1RM. |
Rodrigues et al. [23] | Investigate the acute effect of three different warm-up protocols on a maximal isokinetic strength test. | 22 males | 3 protocols:
| None of warm-ups were able to change the total work of maximal isokinetic strength. |
Mina et al. [24] | Examine the influence of another form of variable resistance during a warm-up on subsequent free-weight 1RM back squat performance compared to free-weight resistance alone. | 16 males | 2 protocols:
| The results are indicative of a potentiating effect of chain-loaded resistance in a warm-up. |
Ribeiro and Romanzini [25] | Investigate the acute effect of different warm-up procedures on the repetition performance of a fatiguing resistance training protocol designed to induce metabolic stress. | 15 males | 4 protocols:
| No significant difference for the sum of repetitions or for fatigue index among conditions for the 3 exercises. |
Abad et al. [26] | Investigate whether the combination of a general with a specific warm-up protocol would improve leg press 1RM values compared with a specific warm- up protocol. | 13 males | 2 protocols:
| These results suggest that a general with a specific warm-up protocol induced temperature-dependent neuromuscular adjustments that increased muscle force production capacity. |
Chattong et al. [27] | Investigate the potentiating effects of different levels of external resistance during box jumps on vertical jump performance. | 12 males | 5 protocols:
| Performing an active dynamic warm-up with or without a weighted vest produced significantly greater posttest vertical jump performance. |
Sotiropoulos et al. [28] | Determine the effects of a specific warm-up using half-squats at low and moderate intensity on vertical jump performance and electromyographic activity of the thigh muscles. | 26 males | 2 protocols:
| The use of a specific warm-up that includes half-squats performed explosively with low to moderate intensity, improves countermovement jump performance. |
Barroso et al. [29] | Investigate the effect of different intensities and durations of general warm-up on 1RM performance. | 16 males | 5 protocols:
| Long-duration low-intensity general warm-up seems to be appropriate to improve 1RM performance in strength-trained individuals |
Resende et al. [30] | Analyze different types of warm-up on the physical performance of Paralympic powerlifting athletes. | 12 males | 3 protocols:
| The different types of warm-up methods did not seem to provide significant differences in the force indicators in elite Paralympic powerlifting athletes. |
Girard et al. [31] | Investigate the influence of two warm-up protocols on neural and contractile parameters of knee extensors | 10 males | 2 protocols:
| Running and strength-based warm-ups induce a similar increase in knee extensors force-generating capacity by improving muscle activation. |
Main characteristics of studies.
It has been evidenced that the warm-up brings positive effects to the subsequent physical exercise, so it is very important to study it and understand how it can be manipulated according to the specificity of exercise training and performance. The purpose of this investigation aimed to analyze and discuss the main results of the literature regarding the effects of warm-up on force production, as well as to analyze those responses during resistance training and maximal strength assessments. The scarcity of research on warm-up protocols in resistance training and strength performance is notorious. Nevertheless, it is possible to verify that the results obtained in most studies are positive. The use of warm-up causes enhancement of performance when external loads are used, especially when the intensity is high. However, more research should be carried out on this topic.
The selected articles of this review tend to focus on the effects that warm-up produces on resistance training, namely the effects on strength performance. Due to the scarcity of articles about the addressed issue, it was difficult to compare the different types and approaches of warm-ups. Moreover, it was also noticed that exercises were not the same in different studies, which also difficult outcomes analysis.
In the study of Rodrigues and collaborators, [23] three different types of warm-up were compared: a general warm-up, a specific warm-up, and a warm-up through stretching, to understand whether it would influence maximal isokinetic training. The results reported that the three types of warm-up had no adverse effect or any type of improvement in acute muscle strength. However, it is important to highlight that the peak of concentric torque reported a lower value in the specific warm-up protocol when compared to the control group, which may mean that the use of a specific warm-up tends to reveal positive results when compared to the control group, which did not perform any type of warm-up.
The study by Ribeiro and Romanzini [25] aimed to compare the effects of three types of warm-ups on the performance of resistance training: a specific warm-up, an aerobic warm-up, and a combined warm-up of both, also using a control group. This study had the particularity of evaluating the performance using repetitions until failure, thus observing whether the conditions would affect resistance training. As in the abovementioned study, none of these conditions showed significant differences in resistance training, although no negative effects were observed after applying a warm-up. This study seems to suggest that the warm-up protocols used do not contribute to the development of strength.
Although the previous two studies have not found a significantly positive response in the strength assessment, the literature reported several benefits on the human body when considering the implementation of warm-up before resistance training [12]. In the study by Abad and his collaborators, [26] the aim was to understand if the implementation of a general warm-up before a specific warm-up would be beneficial when compared with a specific warm-up only. The tests were measured in the leg-press exercise, being evaluated by its 1RM. Two protocols were used in order to determine the effects of warm-up on training: combined warm-up (general with specific) and specific warm-up. The results of the combined warm-up were higher values in the force production, in comparison with the specific warm-up. Considering the positive results of the tests, a combined warm-up would have more benefits than a specific warm-up.
Barroso and his collaborators, [29] purposed to understand the best intensity to use in the general warm-up before the specific warm-up. Thus, four combined warm-up protocols were performed, with different intensities and durations, which were compared with each other and with a control group, which performed only a specific warm-up, with no general warm-up. These researchers found that a general warm-up followed by a specific warm-up would be more beneficial to increase the strength when compared to a specific warm-up that reported lower results. The authors also suggested that when performing the aerobic component of general warm-up, it should be long lasting with low intensity for better results.
On the other hand, the results of Krzysztofik and Wilk [22] did not corroborate with the studies of Abad and his collaborators [26] and Barroso and his collaborators [29]. This study aimed to compare a specific warm-up (named conditioning activity) with a general warm-up, performed before a bench press exercise. In this study, the specific warm-up was performed with a different exercise (plyometric push-ups) from the training exercise (in this case, bench press). The researchers concluded that the use of a specific warm-up had significant results in the strength assessment compared to the general warm-up. The outcomes showed improvement in the bench press exercise performance, being incongruent with the previously mentioned results.
Otherwise, Ribeiro and colleagues, [21] aimed to understand the best intensity to use when only a specific warm-up was performed. This study verified if three types of warm-ups would have an effect on strength training, and its protocols would use three types of external loads in the warm-up exercise: 40% of the training load, 80% of the training load, and the combination of the 40% and 80% of the training load. Positive effects in force production were found when warming-up with higher loads (80% of the training load) before the squat exercise training. The same authors also reported that, when performing a warm-up with low loads and repetitions, there was no effect on strength training performance.
Similar to Ribeiro and colleagues, [21] Minas and collaborators [24] evaluated a specific warm-up in their study. These authors, although also used the squat exercise in their study, used two different warm-ups. A protocol with a chain-loaded (as external weight) and another where it would be used only with the weight of the body, without any help from external loads. The aim of this study was to perceive the effect of another form of variable resistance in a warm-up compared to a warm-up with only bodyweight. After applied the two defined protocols, it was concluded that using a chain-loaded weight as the body’s external weight can enhance our results in the field of strength. Then, it is possible to notice that this study is in agreement with the previous one, although it cannot be directly compared. Both concluded that when using a warm-up with external loads to our body, the results tend to be better.
Sotiropoulos and his collaborators [28] carried out a study to determine the effects of a specific warm-up using low and moderate-intensity squats in the vertical jump. In his study, two warm-up protocols were performed using two different external loads (low and moderate), before performing the countermovement jump. Both protocols demonstrated to be effective when performed before the vertical jump, reporting significant results in the acute force production and the electromyographic activity, showing to be quite beneficial for the countermovement jump.
In the study of Chattong and his companions, [27] which aimed to investigate the potentiating effects of different levels of external resistance (weight vest) during box jumps in the vertical jump, five different warm-ups were assessed. The control condition was performed without any external load and then, the experimental warm-ups were performed with additional weight from 5% to 20% of bodyweight. In this study, the researchers concluded that no improvements in force production were found when increasing the load in the different warm-ups, not even any between using a vest or without it.
The study by Resende and his collaborators [30] aimed to analyze three different types of protocols, to understand their effect on the physical performance of paralympic powerlifting athletes. The protocols applied were: without warm-up, traditional warm-up and stretching warm-up. The results indicated by the researchers revealed that there were no significant differences when applying any of the protocols studied. Although the results did not show significant results, it is important to note that the participants were highly trained athletes and this might have triggered these results.
Gerard and his colleagues, [31] performed the following protocols: running-based warm-up and strength-based warm-up to investigate the influence of two warm-up protocols on the neural and contractile parameters of the knee extensors. It was revealed a significant shortening of time to contract, while the other twitch parameters did not change significantly. Thus, they concluded that both protocols can influence strength training and muscle contraction during training.
After analyzing these studies, there is still controversy around the issue of warming. All studies included in this narrative review are relatively recent, but a consensus has not yet been reached, neither what is the best type of warming up that will have the best results on force production and strength performance. Further investigations should be developed to provide a consensus and clarify the subject. For future studies, it would be interesting to explore this topic a little more. We suggest to study the effect of warm-up on strength training, exploring different types of intensities, in order to achieve more robust and concrete results. The same studies should not be based on a single exercise, but rather deepen the study on several exercises performed in a sequence. It is true that, in a real training context, no single exercise is performed. For example, most resistance training includes more than one exercise and for different muscular groups. It would also be interesting to verify if it will be necessary to warm-up before each exercise and specifically for each exercise, or if the first warm-up before training is enough to guaranty better results during the entire training.
With this narrative review, we could verify that there is a great lack of studies on the subject of warm-up for strength performance, resistance training performance, and force production. It was also possible to show that some authors did not report benefits after warm-up, however, others found quite significant results in their studies. These positive results were either after using only a specific warm-up or using a general warm-up followed by a specific warm-up. So, it is possible to determine that special attention on this topic is needed. Nevertheless, most of the studies tended to suggest that a warm-up should be performed before resistance training is performed. The increased strength outcomes seemed to be better when a higher load is used during warm-up, with few repetitions. Moreover, the use of a general warm-up showed to be beneficial in some specific assessments. Therefore it can also be a strategy to be applied and combined with the specific warm-up. Further investigations should be developed to better understand and determine the effects of warm-up, or even other studies using another type of exercise, so we can provide a more in-depth conclusion.
This work was supported by national funding through the Portuguese Foundation for Science and Technology, I.P., under project UIDB/04045/2020.
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