http://www.revistadechimie.ro REV.CHIM.(Bucharest)♦ 69♦ No. 2 ♦ 2018310

Optimised Pu-erh Tea Infusion by Experimental Designand Response Surface Methodology

RALUCA DANIELA ISOPESCU1, ANA MARIA JOSCEANU1*, IULIAN ILIE MINCA2, GABRIELA ISOPENCU1

1University Politehnica of Bucharest, Faculty of Applied Chemistry and Material Sciences, 1-5 Polizu Str., 060021, Bucharest,Romania2National Research and Development Institute for Chemistry and Petrochemistry - ICECHIM, ANALYSIS, TESTS & TESTINGDepartment, 202 Spaiul Independentei, 060021, Bucharest, Romania

Apart from the green and black tea consumption, post-fermented Pu-erh specialties are increasingly presenteven on emerging markets as the one in Romania. Despite simplicity, infusion preparation should beconducted so that polyphenols content reaches its peak, and toxic elements (heavy metals, nitrates) areeliminated or limited to their maximum admitted levels for foodstuff. The study was carried out based on acentral composite design, infusion composition being used for proposing regression models representingthe response surface generated by the measured parameters as function of working conditions. The analyzedfactors were contact time, temperature, and stirring rate. Multi-objective optimization signaled series ofoperational parameters values yielding maximum polyphenols, at minimum cadmium and nitrate contentsin Pu-erh tea infusions. Typically, infusions prepared at 82 - 83°C, using 6 min contact time, and 170 rpmcontain 8.9 % polyphenols, while cadmium reaches 3.98 µg/mL, and nitrate 0.35 mg/mL.

Keywords: Pu-erh, optimization by multi-objective tools, polyphenols, cadmium, nitrate

Camellia sinensis, with its assamica and sinensisvarieties, is an amazing example of turning leaves of oneplant into a series of products bringing a 31.2 billion USDworth market in 2016 [1]. This is the value of sales of allforms of tea (leaves, instant, liquid concentrate, and readyto drink beverages, all considered at the selling price of themanufacturer).

The long-time habit of tea drinking has developed into acontinuous quest for natural resources of antioxidants, anti-inflammatory, tumour blocking, and weight control agents.With more than 4000 years of history, tea is presently tradedas non-fermented (white and green), semi-fermented(oolong), fully-fermented (black) and post-fermented(Chinese dark) tea in over 160 countries. Post-fermentedChinese teas were traditionally produced from the broad-leaf variety, assamica, in the South-Western China andcarried on horseback to Tibet, Xinjiang. The fungi inducedoxidation is responsible for the chemical transformationsduring the post-fermentation process, the strains involvedbeing Aspergillus, Penicinillium, and Saccharomyces genus[2]. The natural microbiota isolated from Pu-erh teas,namely Aspergillus (A. fumigatus, A. marvanovae)Rhizomucor (R. pusillus, R. auricus), and Candida mogiihave been successfully tested for controlled inducedfermentation [3].

Traditional post-fermentation does not involve artificialplacement of microorganisms into the piled raw tea. They arenaturally originating from the air and water present in situ,during the horseback trip along the famous Chinese Tea-horseRoad [4]. Nowadays Pu-erh is a designated origin productfrom 11 cities, 75 counties, and 639 townships in Yunnanprovince, situated namely between parallels 21°10’ and 26°22’north latitude and 97°31’ and 105°38’ east longitude.

Chinese dark teas have proven their efficiency inreducing inflammations and boosting the immune system[5-7]. The first paper on the lipid lowering effect dates backin 1986 [8], being followed by other comprehensive trials* email: a_josceanu@chim.upb.ro

[9,10]. The Southeast Asian market has increasedsignificantly, so the Pu-erh tea output reached 99,000 tonsin 2007, raising more than 58 % of the Chinese dark teasmanufactured [11]. Starting with 2010 Pu-erh tea, bothraw and ripen, has become available on the Romanianmarket, mainly in specialized tea shop.

Technological processing and formulations give raise tonumerous differences in the chemical composition [12,13]and biological effects of prepared infusions. Liebert et al. [14]paid attention to the total polyphenols content and Troloxequivalent antioxidant activity in green and black teas infusions.Effect of shooting period and processing system factors onthe extract content in terms of caffeine and crude fibre ofblack tea has been studied by Ozdemir et al. [15]. They provedby one factor analysis that caffeine content was not affectedby the processing route, but was highly dependent on theshooting period and the intervals between shootings.Castiglioni et al [16] investigated the antioxidant propertiesand sensory attributes of several white and green teasinfusions varying steeping temperature, time, and particlessize. Their one factor study pointed out that the maximumextraction efficiency in terms of total phenolics and flavonoidsoccurred when using cold water for 120 min and 90°C waterfor 7 min.

Whenever two or more factors may influence theresponse of a system, scientists have relied on ANOVA[17] and/or response surface methodology (RSM) andregression to optimize processes and composition ofproducts [18-20]. Similar approaches have been taken inincreasing the rutin yield extracted from mate tea beverages[21]. The content of chlorogenic acid, rutin, and antioxidantactivity in green teas infusions have been optimized by RSMby central composite design [22]. Time and temperature havebeen the chosen independent variables, the variation rangesmatching the European preparation habits. Optimizedconditions of 94.15°C and 14.91 min lead to maximumantioxidant activity of infusions.

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Infusions prepared from leaves and stems of yerba matetea were also subjected to central composite designexperiments to identify the proper contact time, temperature,and water volume leading to the highest extraction yields offour chlorogenic acids [23]. Infusing 2 g of mate tea in 300 mLof water at 95°C, for 16 min lead to the maximum chlorogenicacids content.

Regular consumers do not have access to antioxidant,anti-tumoral capacity assay or chemical compositiondetails, but they do have access to chronometers andkitchen thermometers, so they could easily accede to aposition of control when it comes to health and nutrition.There is an increased interest for accessible andrecommendable infusing strategies that deliver Pu-erh teainfusions containing the highest possible levels of beneficialspecies, while keeping harmful entities at bay. The presentstudy draws attention to the composition of Pu-erh tea infusionsin terms of polyphenols and inorganic contaminants content,and aims to determine to what extent they are affected by theextraction conditions. Since most published optimisationstudies have been focused on increasing the yield incompounds of interest, the present approach of maximizingthe total polyphenols content, while minimizing the levels ofcertain contaminants represents a practical investigationpathway, delivering sensitive information to specializedaudience and consumers. A set of optimal conditions forpreparing Pu-erh tea infusions are identified using the RSMmethod and multi-objective optimisation tools.

Experimental partSamples

A cooked Pu-erh tea harvested in 2010 and available onthe Romanian market was selected for the factorialexperiment studies. The loose Pu-erh tea was a Jing Gufactory product, in the Simao, Yunnan province, China, abest quality product (gong ting grade) prepared from smalltippy leaves of assamica variety of Camelia Sinensis. Itwas stored in the original paper bag in plastic sealedcontainers at 4°C in the refrigerator. Another 3 cooked and3 raw Pu-erh samples harvested in 2007-2010, availableon the Romanian market, were also selected to determinethe infusions content prepared according to the optimizedconditions.

ReagentsAn ACS standard of gallic acid (97.9% content, Fluka)

was used for calibration purposes in the total polyphenolscontent determination. Methanol (99.9% content, Sigma-Aldrich) was used for the extraction of polyphenols fromtea leaves without further purification. Stock standardsolutions for ion chromatography (Fluka), of certified 1000mg L 1 Pb, Cd, Mn, Co, Ni, and Zn concentration traceable toSRM NIST were used to prepare calibration solutions byappropriate dilution with deionized water. A certifiedcombined stock standard solution for ion chromatography(Merck), traceable to SRM NIST, and containing F- (100 mg L1), Cl-, Br- (250 mg L 1), NO3

-, SO42- (500 mg L 1), PO4

3- (1000mg L 1) was used for calibration purposes in thequantification of anionic species present in the tea infusions.

All calibration standard solutions were prepared by dilutingthe stocks appropriately, just before use. All other reagent usedfor quantification purposes were analytical grade, with a 99.9%content or better. All volumetric glassware used for thepreparation of standard solutions was A class. Ultrapure water,18.2 MΩ cm 1, filtered through a 0.20 µm pore membrane,produced by an Easy Pure Rodi Branstead system (Milli-QGradient A10, Millipore) was checked for the anion and heavymetal content, and used for preparing standard solutions and

tea infusions.

Infusions preparationThe experimental study was carried out using a full

factorial scheme for a three investigation of the workingconditions. The analysed factors were contact time,temperature, and stirring rate employed for infusionpreparation: 2-15 min, at 65- 85°C, and 0-150 rpm. Carefullyweighed grounded leaves, 1.0000 g load, were transferredinto glass containers with screwed plastic lid, and furthercontacted with 80 mL deionized water. Both tea leavesand water were preheated at the temperature required foreach experiment. The time was recorded from themoment tea and water were put in contact. Followingextraction, leaves were separated from the aqueous phaseby filtration, using medium size pore quantitative filteringpaper; the aqueous extracts were stored into glasscontainers with screwed plastic lid.

Infusions analysisTotal polyphenols content

After cooling at room temperature, 1 mL aliquots weresampled from each infusion, diluted to 100 mL with deionizedwater and tested for the total polyphenols content using theFollin-Ciocalteau reagent, and 30 min reaction time.Polyphenols content was expressed as g gallic acid/100 ginfused tea leaves, using a 5-40µg mL 1 concentrationcalibration range [24]. The calibration curve for gallic acidpresented slope, b, and intercept, a, equal to 0.01238, and0.0151µg mL 1 respectively, standard error of slope, sb, of 4 .10-5µg mL-1, intercept standard error, sa, of 8 . 10-4µg mL-1,determination coefficient, R2, of 0.9998, and 0.0024 responsestandard error, yS. The method detection limit (LOD) was 0.03µg mL-1, while the quantification limit (LOQ) was 0.09 µgmL-1; it had a 0.9% relative standard deviation (RSD) whenassessing repeatability. Samples and standards were run astriplicates.

Analysis of the ionic contentThe anions (NO3

-, NO2-, I-, Br-, F-, SO4

2-, and PO43-) and heavy

metals (lead, cadmium, cobalt, nickel, and zinc) contentswere evaluated by ion-chromatography (IC). Prior injection inthe chromatographic systems, all freshly prepared infusionswere filtered through a battery of dedicated filters for theremoval of phenols, anthocyanes, azo dyes, aromaticcarboxylic acids and aldehydes (OnGuard P II cartridge),tensioactive agents and lipids (OnGuard II RP cartridge), andparticulates (0.45 µm cellulose membrane). Filtered infusionswere frozen at -16°C and stored before IC analyses, eachsample in triplicate, within 24 hours after processing.

Analysis of anions was carried out at 30°C, on a ICS 3000Dionex system equipped with a IonPac AS22 (4 . 250 mm)separation column, and a conductivity detector, using 20µLvolume samples. The mobile phase (0.0045 M Na2CO3 and0.0014 M NaHCO3) was delivered at 1.0 mL min-1 flow rate.The experimental parameters allowed retention timesbetween 3.92 min (F ) and 14.60 min for SO4

-2, characterizedby 0.19 - 0.73% RDS. Anions calibrations were carried out inthe 0.02 - 10 mg L-1 range for F , 0.1-100 mg L-1 for PO4

-3 , and0.1-50 mg L -1 for the remaining 6 ions. Slopes varied from0.069 to 0.323 mg L-1, with standard deviations of 0.04 to 0.141mg L-1, intercepts not higher than 0.004 mg L-1, R2 in the 0.9991– 0.9998 range, and yS less than 4.9 mg L-1. Repeatability fora 2 mg L-1 F-, 10 mg L-1 Cl-, NO2

-, Br-, NO3- and SO4

2-, and 20 mgL-1 PO4

3- solution was 0.38% in the case of F-, 0.23% for Cl-,0.26% for NO2

-, 0.19% for Br-, 0.14% for NO3-, 0.04% for PO4

3-

and 0.25% for SO42-.

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Analysis of heavy metals was run at 30C, on a ICS 3000Dionex system equipped with post column derivatizationand spectrophotometric detection at 530 nm, workingisocratically with IonPac CS5A (4´250 mm). The eluentflow rate was 1.2 mL min-1, the derivatization reagent flowwas 0.6 mL min-1, for 100 µL injection volume, and 375-µLmixing knitted reaction coil. The mobile phase containedoxalic acid (0.080 M), tetramethyl ammonium hydroxide(0.100 M), and potassium hydroxide (0.050 M). The postcolumn derivatization reagent consisted of p-aminoazoresorcinol (PAR, 0.25 mM) dissolved in 2-dimethylaminoethanol (1.0 M), ammonia (0.50 M), and sodium acidiccarbonate (0.30 M). The average retention times variedfrom 2.65 min, for Pb(II), to 9.01 for Ni(II), with RSD lessthan 0.8%. Calibrations were carried out in the 2 - 100 µgL-1 range, giving linear regressions for all six metals in termsof peak area, with b values of 0.03 -0.27 µg L-1, sb equal to0.01- 0.13 µg L-1, 0.1- 9.1 µg L-1 a values, sa of 0.36 - 3.93 µgL-1, and R2 in the 0.9997- 0.9999 range. 10 replicatesrepeatability tests at the calibration ranges limits gave 0.19- 3.81% RSD, with higher values for lower levels of copperand zinc.

Polyphenols in tea leavesThe polyphenols levels in the Pu-erh leaves were also

determined by means of Follin-Ciocalteu reagent in highlybasic solutions, following extraction into 70 % methanol[24]. 0.2000 g finely grounded leaves were extracted twicewith 5 mL hot methanol on water bath. After centrifugationand cooling to room temperature, the combined extractswere brought to 10 mL with cold water-methanol mixtureand diluted 1:100 with deionized water. The polyphenolsquantification in tea leaves followed the procedure usedwhen assessing infusions, the result being expressed as ggallic acid/100 g dry tea leaves.

Statistical modelling and optimizationThe relative importance of the operating parameters

duration of extraction, temperature, and stirring rate uponthe polyphenols and ions content in the tea infusion wasevaluated according to a 23 factorial experimental design.The central point corresponds to a contact time of 6 min,at 75°C, and 100 rpm. Coded variables were used in thestatistical modelling, +1 and -1 for the high and lowerlevels respectively, and 0 for the centre point. The 4replicates in the centre of the experimental design gavethe experimental error.

Modelling and optimisation of tea infusion content werecarried out in the frame of RSM Second degree polynomialmodels were proposed, including the interaction of purequadratic terms, with possible maximum/minimum valuesin the range of factor variation,

(1)

where y is the computed value for polyphenols or the mainionic species.

The experimental design was augmented by six stars pointsaccording to the orthogonal central composite design [25];the star points laid at α distance of 1.41 from the central point,value calculated according to Myers and Montgomery [26]:

(2)

where F is the number of factorial points (F = 8), k thenumber of factors (k = 3) and n0 the number of centralpoints (n0 = 4). The coded variables for the experimentalprogramme are collected in table 1.

Using the regression equations describing the extractionprocess, the optimization of operating parameters wascarried out with the declared objective to maximize thepolyphenols content and minimize the toxic elementsconcentration. Optimal operating conditions wereidentified by applying multi-objective optimizationprocedures implemented in MatlabTM.

Results and discussionsThe experimental results obtained in the 23 full factorial

programme are presented in table 2. All extractions in thisexperiment were carried out using the same loose cookedPu-erh tea product, containing 10.2% polyphenols in theleaves. The experiments in the factorial points were usedto calculate the influence of x1, x2, x3 factors upon themeasured infusions content. The experimental error,expressed as standard deviation, is accessible from datarecorded in the centre point.

All 23 factorial data were used to rapidly scan the factorsinfluence upon infusions content, by determining thevariation of total polyphenols, heavy metals, and anionsconcentrations when the coded factors spanned between-1 and +1 [25]. Denoting by y the measured componentconcentration, the main effect is defined as the y+ - y-difference, y+ representing the mean y value at maximumfactor level, and y- the corresponding measured mean valueat minimum factor level (table 2). Results show that thepolyphenols content is influenced by all three factors, itsvariation at high and low parameters values beingsignificantly larger than the experimental error. Thetemperature increase exerts the highest influence uponthe polyphenols content. Longer extraction time and ahigher stirring rate are also accompanied by larger levelsof polyphenols. The cadmium present in tea infusion is ofmajor concern, even if the values recorded are lower thanthe maximum admitted level in foodstuff of 50 µg L-1 [27].The Cd concentration decreases with increasingtemperature and decreasing contact time, and it is notaffected by the stirring rate. Pb was rarely detected, so itwas no further considered in the analysis. Zn is also presentin the tea infusion in small amounts, being known for itsextremely reduced toxicity for humans [28]. Itsconcentration variation in the factors variation range is of

Table 1 EXPERIMENTAL AND CODED VARIABLES

IN THE 23 FACTORIAL PROGRAME

^

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similar order of magnitude as the experimental error, sothe operating parameters modification failed to significantlyaffect Zn extraction in the tea infusion. Therefore, it wasno longer considered in the optimization process. Theanions present in the samples are less influenced by thevariation of extraction conditions than polyphenols and Cd,but given the toxicity of NO3

-, whose concentration isslightly influenced by the duration and stirring rate, theidentification of optimum infusion conditions also includedthe nitrate content. The experimental data in the star pointswere added to the full factorial data, (table 3) containingthe experimental basis of the optimization process.

The coefficients of second degree polynomials werecalculated with the regression tool implemented in EXCEL.Since not all regression coefficients were significant at a5% significance level, a selective model reduction wasapplied, by neglecting the less significant coefficients.Reduction was carried out by keeping the determinationcoefficient at an acceptable value, while preserving theextraction meaningful factors. The lack of fit test wasperformed for the final reduced models, validating theusage of the regression models for the subsequentoptimization step.

(3)According to ANOVA results for polyphenols content

(table 4), the model in equation (3) is significant andexplains 76.2 % of the experimental variability, as R2 =0.762. All coefficients considered in the reduced modelare significant at a 5% level, except for b3. The term referring

to the influence of x3 (agitation rate) was neverthelessmaintained in the model since its influence upon thepolyphenols content was significant compared to theexperimental error (table 2). The lack of fit test showedthat the computed F-statistics value (5.77) is lower thanthe critical F value at 5% significance (F9,3 = 8.81), andascertained that the model can be further used inrepresenting the variation of polyphenols concentration inthe range of studied operating parameters.

(4)

The determination coefficient for cadmium model inequation (4), R2, is 0.823, and the Fisher test for modelsignificance returned a p-value of 8.1E-5 (table 5). The lackof fit test proved that the model can be trusted for theprediction of Cd concentration in tea infusions, as thecomputed F-statistics (8.15) is lower than the critical valueF10,3 (8.78).

(5)

As for the NO3- ions, the model proposed by equation (5)

retains only a linear combination of contact time andmixing rate (table 6), R2 being 0.928. The lack of fit test,reveals a computed F-statistic (0.23) lower than the criticalF12,3 value (8.74), and proves that the proposed model issuitable for further optimization step.

The response surfaces using the polyphenols, cadmium,and nitrate regression models are shown in figure 1. Thepolyphenols content reaches a maximum value within thetemperature and time variation ranges, and constantly

Table 2COMPOSITION OF TEA INFUSIONS PREPARED IN THE 23 FACTORIAL PROGRAME

Table 3FULL EXPERIMENTAL DESIGN PROGRAME

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increases with agitation speed, as only the linear term in x3was retained in the model. The cadmium content does notdepend on the mixing rate, and presents a minimum in theduration and temperature ranges investigated. The extractednitrate increases with duration and agitation rate, but it is notinfluenced by temperature.

Several approaches were considered when seeking theoptimum conditions for tea infusion preparation. A first goalis to maximize the polyphenols content, since theantioxidant properties strongly recommend it. As anyoxidation state of Cd induces toxic effects in humans, itspresence in any tea infusion should be kept well below themaximum admitted limit. Consequently, the maximizationof polyphenols and minimization of Cd concentrations wereconsidered. Since the maximum of a function is unlikelyto correspond to the minimum of the second, a multi-objective technique was applied where a trade-offbetween the criteria is accepted. This leads to a set ofoptimal solutions represented by the Pareto front, whereone objective can be improved only by the expense ofanother objective. The objective function for simultaneous

optimisation of two criteria is a vector,F(x1, x2, x3)=[f1(x1, x2, x3),f2(x1, x2, x3)]. A minimisationproblem was considered, min F(x1, x2, x3), and, with theobjective vector components defined as:

The optimization was performed with the gamultiobjfunction implemented in Matlab. A series of equally optimalsolutions was obtained, suggestively presented by thePareto front in figure 2(a). The Pareto front provides theoptimal solution, corresponding to a Cd content varying inthe 3.90 - 4.71 µg L-1 range and polyphenols concentrationfrom 8.65 to 9.10 %. Several operating parameterscorresponding to the Pareto front are available in table 7,accompanied by the NO3

- composition, calculated usingthe proposed model for the x1 and x3 values from the Paretofront. Operating conditions were identified, where thepolyphenols content is high and Cd concentration varies

Table 4NOVA RESULTS FOR THE POLYNOMIAL

REGRESSION MODEL PROPOSED FOR THEPOLYPHENOLS CONTENT IN TEA INFUSIONS

Table 5ANOVA RESULTS FOR THE POLYNOMIAL

REGRESSION MODEL PROPOSED FOR THE CDCONTENT IN TEA INFUSIONS

Table 6ANOVA RESULTS FOR THE POLYNOMIAL

REGRESSION MODEL REFERRING TO NO3

CONTENT IN TEA INFUSIONS

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Fig. 1. Response surfaces forpolyphenols, Cd, and NO3

- levels in Pu-erh tea infusions based on the proposedmodels: a) surface plot for polyphenolsat x3 = 0; b) surface plot for polyphenolsat x2 = 0; c) surface plot for Cd at x3 = 0;

d) surface plot for NO3 at x2 = 0.

Table 7 OPERATING PARAMETERS AND TEAINFUSIONS CONTENT FOR SEVERALOPTIMAL OPERATING CONDITIONS

FROM PARETO FRONT CONSIDERING ATWO CRITERIA-VECTOR OBJECTIVE

FUNCTION

Fig. 2. Pareto front for multipleobjectives function: a)

Polyphenols - Cd front; b)Polyphenols - NO3

front.

Table 8OPERATING PARAMETERS ANDTEA INFUSIONS CONTENT FOR

A SERIES OF OPTIMALOPERATING CONDITIONS

FROM PARETO FRONTCONSIDERING A THREE

CRITERIA-VECTOR OBJECTIVEFUNCTION

CONDITIONS

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around 4 µg L-1, a value close to the admissible limit fordrinking water.

If the optimisation problem includes the minimisation ofNO3 content in tea infusions, the vectorial objective functionshould consist of three objectives: maximisation ofpolyphenols and minimisation of Cd and NO3

- levels, as inequation (6). The optimisation procedure results are presentedin figure 2(b). The operating conditions corresponding to aseries of Pareto front solutions are given in table 8.

Min F(x1, x2, x3) =[f1(x1, x2, x3), f2(x1, x2, x3), f3(x1, x2, x3)] (6)

where

This combined information signals that the decrease inNO3

- content is achieved on the expense of a severedecrease of polyphenols concentration in the tea infusions.If extraction occurs at very low agitation rates, the NO3concentration decreases significantly, but the polyphenolscontent does not exceed 6-7%. Considering that the cookedPu-erh leaves contained initially 10.2% polyphenols, 7%polyphenols in the tea infusion corresponds to a polyphenolsin the infusion / polyphenols in the leaves ratio, PPhinf/PPhleaves, of approximately 68%. As the main goal of thepresent study is an increased polyphenols extraction andlimited contaminants concentrations in infusions, theoptimum operating parameters considered are:temperatures between 82 and 83°C, 6 min contact time,under intense agitation, 170 rpm. In these conditions, the PPhinf/PPhleaves is 87%, Cd concentration below 4 µg L-1 and NO3

-

concentration is acceptable, 0.35 mg/mL.The optimised operating conditions were further tested in

preparing infusions using another 6 raw and cooked Pu-erhtea samples, purchased from various suppliers on theRomanian market, all results being collected in table 9. The 0labelled line corresponds to the Pu-erh tea used in theexperimental program carried out for the statisticalmodelling and optimization purposes. It is followed by theresults obtained for another 3 cooked tea types, harvestedin 2007 and 2008. The last three refer to raw Pu-erh samplescharacterized by lower polyphenols levels both in leaves,and in the prepared infusions. The PPhinfusion/PPhleaves ratio,a measure of the extraction efficiency, is also reported. Allinvestigated samples validated the set of operatingconditions identified during the optimization step, in termsof polyphenols and contaminants content.

ConclusionsThe influence of temperature, contact duration, and

agitation rate on the quality of infusions prepared from cookedPu-erh tea was studied using response surface methodologyand multi-objective optimization. Infusions werecharacterized by the total polyphenols content and levels of

Table 9 TEA INFUSION POLYPHENOLS, CD,AND NO3

CONTENTS IN DIFFERENTPU-ERH INFUSIONS PROCESSED ATOPTIMUM DECLARED OPERATING

heavy metals and major anions. Statistical modelling wascarried out for the species undergoing significant concentrationchanges within the investigated range of the extractionparameters. The proposed models for the variation of totalpolyphenols, cadmium, and nitrate levels were statisticallyvalidated, and further used in optimization. Maximization ofpolyphenols and minimization of cadmium and nitrate occursat 82-83°C, 6 min contact time, under 170 rpm agitation,selected from a series of identified optimal solutions. This setof working parameters was validated for 6 other Pu-erhsamples of various harvesting time and distributors fromYunnan province, China.

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Manuscript received: 5.07.2017