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165I. JELII et al.: Microbiological and sensory quality of rennet cheese whey, Mljekarstvo 62 (3), 165-178 (2012)

*Corresponding author/Dopisni autor: Phone/Tel.: +385 (0)1 4605 024; E-mail: [email protected]

Original scientific paper - Izvorni znanstveni rad UDK: 637.344

Influence and comparison of thermal, ultrasonic and thermo-sonic

treatments on microbiological quality and sensory properties of

rennet cheese whey

Irena Jelii1, Rajka Boani1, Mladen Brni2, Branko Tripalo3

University of Zagreb, Faculty of Food Technology and Biotechnology, 1Department of Food Engineering,Laboratory for Technology of Milk and Dairy Products, 2Department of Process Engineering,

Laboratory for thermodynamics, R&D Centre for Ultrasound Applications in Food Technology and Bio-technology, 3Department of Process Engineering, Laboratory for Unit Operations,

Pierottijeva 6, 10000 Zagreb, Croatia

Received - Prispjelo: 02.07.2012.Accepted - Prihvaeno: 21.08.2012.

Summary

Ultrasonication and thermo-sonication belong to alternative, non-thermal food processingmethods. The aim of this study was to investigate the influence of different ultrasound power inputs(240 W, 320 W, 400 W) without and in combination with heat pre-treatment on microbial inactiva-tion and sensory properties of rennet cheese whey in comparison with conventional pasteurizationbatch processes. Ultrasonication treatments had no impact on reduction of any group of studied mi-croorganisms. Microbial inactivation caused by thermo-sonication treatments with pre-heating to 35C or 45 C increased with nominal power input and/or exposure times and was probably due to theheat improved ultrasonic cavitation. Thermo-sonication treatments at nominal power input (400 W)

and preheating to 55 C were the most effective resulting in greater microbial reduction compared tothat observed by simulating pasteurization processes, but occurred probably due to developed heatsolely. Sensory properties after ultrasonication and thermo-sonication were considerably improvedin comparison with that after simulated pasteurization processes. Mouth feel of whey samples wasconsiderably better, there was no occurrence of sediment and colour remained unchanged in almostall samples.

Key words: ultrasonication, thermo-sonication, sensory properties, microbial inactivation,rennet cheese whey

Introduction

Whey is a by-product of cheese production.Liquid whey consists of approximately 93 % water,4.5 % lactose and 1 % proteins. In smaller amountsminerals, vitamins and milk fat are also present(Jelii et al., 2008).

Due to a high content of essential aminoacids, whey proteins have a much higher biologicaland nutritional value than other proteins of animalorigin. In recent years it has been proved that wheyproteins also show some bioactive effects like anti-microbial, anti-hypertensive and anticarcenogenic

properties (Ree k-Jam brak et al., 2008). Besides,

whey proteins have excellent functional propertiesi.e. foaming ability, solubility, emulsifying properties(Brn i et al., 2008; Jel i i et al. 2008).

However, whey proteins tend to precipitateat temperatures above 60 C (Parris et al., 1991).The high water content and high content of sugarin the dry matter make fresh whey very susceptibleto microbial spoilage so heat treatments, i.e. pas-teurization, are obligatory. As a result of heat treat-ment, sediments of proteins and some salts as wellas redundant sourness are likely to occur (Je lii etal., 2008). In such a manner nutritional and sensory

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166 I. JELII et al.: Microbiological and sensory quality of rennet cheese whey, Mljekarstvo 62 (3), 165-178 (2012)

quality of whey is reduced. Therefore whey is rarelyprocessed into food products like beverages. It ismostly used as animal feed or processed into wheypowder, whey protein concentrates and isolates byapplying processing techniques with high energy

consumption (Brni et al., 2011).In recent years, a lot of effort has been put into

investigation and development of new methods forfood processing that have reduced impact on the nu-tritional content and quality of food, but significant-ly reduce microbial activity. The amount of requiredheat couldtherebybe partially reduced or consider-ably eliminated (Ugar te -Rom er o et al., 2007).

Ultrasonication belongs to this group of methodsand can be defined as sound waves with a frequency

higher than 20 kHz. Ultrasound has a number ofapplications in the food industry which can be divid-ed into applications of low and of high energy ultra-sound. Low energy ultrasound has intensities below1 Wcm-2 and frequencies above 2-3 MHz. It is usedfor non-invasive purposes like foodstuffs characteri-zation, determination of unwanted foreign bodiesin raw materials and final products, level measure-ments in tanks etc. (Reek Jambrak et al., 2010).High energy ultrasound with intensities above1 Wcm-2 andfrequencies between 18 and 100 kHzfound its possible application also in the dairy indus-try for processes like milk homogenisation, clean-ing, enzyme and bacteria inactivation, chymosin and-galactosidase extraction (Reek-Jambrak et al.,2009; Jelii et al., 2010; Bosiljkov et al., 2011).The main active force during ultrasonication is ofmechanical nature and causes the formation and im-plosion of bubbles in a liquid, what is known as cavi-taton (Brn i et al., 2010). During implosions, veryhigh temperatures and pressures occur, reaching up

to 5500 C and 50 MPa, which is believed to be themain bactericidal effect of ultrasound(Villammieland de Jong , 2000; Pi ya sena et al., 2003). That ef-fect is mostly localized; it does not affect a large areaand has not enough potential to destroy most resist-ant spores and bacteria. Consequently ultrasoundtreatments were not accepted as a food preservationmethod (Raso et al., 1998; Pi ya se na et al., 2003).More recent studies report a better lethal effectof ultrasound when combined with heat (thermo-sonication or thermoultrasonication). Many authors

have observed good results in reduction of potentialpathogens number like Escherichia coli (Lee et al.,

2009),Listeria monocytogenes (Pagan et al., 1999;Ugarte-Romero et al., 2007) and Shigella boydii(Ugar te -Rom er o et al., 2007) as well as some non-pathogens likeListeria innocua (Noci et al., 2009;Bermudez-Aguirre et al., 2009). However, those

studies were conducted either on a model solutionsor raw milk where the concentration of the targetmicrobe was known. However, there are no avail-able studies about the impact of thermo-sonicationon the overall microbiological quality of fresh whey.

The aim of this study was to investigate wheth-er thermo-sonificaton could be used as an alterna-tive processing method for the purposes of adequatemicrobiological quality assurance and improving sen-sory properties of rennet cheese whey. Therefore the

influence of different ultrasound power inputs with-out and in combination with heat pre-treatment onmicrobiological quality of whey in comparison withconventional pasteurization processes were evalu-ated. Furthermore, sensory properties i.e. odour,flavour, appearance and colour were compared afterultrasonication, thermo-sonication and conventionalpasteurization processes.

Materials and methods

Whey samplesSamples of fresh unpasteurized sweet whey

were purchased from Vindija d.d. Dairy Industry(Varadin, Croatia) and were stored at tempera-tures below 10 C. The total solids amount wasdetermined by the standard method of drying toconstant mass at 1022 C (Cari et al., 2000).The amount of lactose was determined by a refer-ent method using Na-tungstate and chloramine T inaccordance with the International Dairy Federation(IDF) (Cari et al., 2000). The protein amount was

determined by the Schulz method (Boani et al.,2010), the mineral content by a method accordingto IDF (Cari et al., 2000) and the milk fat contentby Gerbers butirometric method (Boani et al.,2010). Titrable acidity was determined by the Soxh-let Henkel method (Boani et al., 2010).

Storage and thermal treatment of whey samples

After receiving the whey samples from thesupplier, 2 L of untreated rennet cheese whey was

immediately poured into two sterile glass bottles,closed and placed in the refrigerator. Before using it

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for microbiological and sensory analysis, 100 mL ofwhey was poured into a sterile glass vial, stoppered,placed into a thermal controlled water bath withconstant shaking and gradually heated to a roomtemperature (approximately 20 C). The tempera-

ture of the heating bath was maintained at 201 Cto allow heat transfer. The temperature of whey wasmonitored with a thermometer placed into a glassvial.

In addition, for comparison with thermo-soni-cation and ultrasonication, equal volumes of 100 mLof fresh whey were thermally treated at 721 C for20 seconds and at 651 C for 30 min. These par-ticular regimes were chosen to simulate the conven-tional batch pasteurization processes. Heating was

performed in sterile closed glass vials placed into athermal controlled water bath with constant shakingto assure homogenous dispersion of heat inside thesamples throughout treatment. The temperature ofthe heating bath was maintained at 721 C or at651 C to allow heat transfer. The temperatureof whey was monitored with a thermometer placedinto a glass vial. After finishing pasteurization, sam-ples were gradually cooled down to approximately20 C.

Aliquots of each control sample were trans-ferred to test tubes with physiological solution forfurther microbiological analysis. The rest of eachsample was prepared for sensory evaluation as listedbelow. The experiment was conducted in triplicate.

Ultrasonic and thermo-sonic treatments of whey

Fresh whey samples (100 mL) were placed intosterile glasses with a total volume of 300 mL whichwere used as treatment vessels. An ultrasonic device

(Hielscher Ultrasonics GmbH, Germany, modelUP400S, amplitude regulation 20-100 %, frequency24 kHz) with a 7 mm diameter probe was used forthis research. The ultrasonic probe was directly im-mersed in the sample with depth of 1.0 cm fromthe surface. Ultrasonication was performed at 100%, 80 % and 60 % of nominal output power, wherethe power level of 100 % corresponded to approxi-mately 400 W, and the power levels 80 % and 60% corresponded to approximately 320 and 240 W.Exposure times were 5, 6.5 and 8 min whereby the

samples were tempered at room temperature (20C) before each treatment. Thus, there were a total

of 9 ultrasonication treatments, which were denot-ed as 100 %/5 (5 min at power level 100 %), 100%/6.5 (6.5 min at power level 100 %) etc. Thermo-sonication treatments were performed under thesame conditions as mentioned above, only with the

difference of preheating the samples to three differ-ent temperatures (35 C, 45 C, 55 C). Thus, therewas a total of 27 treatments which were denoted as100 %-35 C/5 (5 min. exposure at power level 100% with preheating to 35 C), 100 %-35 C/6.5 (6.5min exposure at power level 100 % with preheat-ing to 35 C) etc. Throughout each treatment, thetemperature of the samples was monitored by an IR-thermometer (Raytek - MiniTemp FS, Raytek, Mel-rose, USA) and recorded in periods of 20 seconds.After each treatment aliquots were transferred totubes with (containing) physiological solution formicrobiological analysis. The rest of each sample wasprepared for sensory evaluation as listed hereafter.Each experiment was performed in triplicate.

Acoustic energy density and ultrasound intensity de-

termination

Acoustic energy density (AED) and ultrasoundintensity (UI) values were determined using a ca-

lorimetric method (Tiwari et al., 2008a; 2008b).Whey samples were sonicated with temperature (T)recorded as a function of time under adiabatic condi-tions using an IR thermometer (Raytek - MiniTempFS, Raytek, Melrose, USA). The initial temperaturerise (dT/dt) was determined by polynomial curvefitting. Since there were no available data on thespecific heat of whey, it was calculated using resultsobserved by chemical composition analysis of whey,as shown in Eq (1):

Cp= wvCpv+ wc Cpc+ wpCpp+ wfCpf+waCpa (1)

wv,

wc,

wp,

wf, w

a= mass contents of the main whey

constituents

Cpv,

Cpc,

Cpf,,

Cpp,

Cpa

= specific heat of the main wheyconstituents (Lelas, 2006).

Ultrasonic power (P), AED (WmL-1) and UI(Wcm-2) values were calculated using Eqs. (2), (3)and (4):

P=m*Cp*(dT/dt)t=0 (2)

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AED = P/V (3)

UI = 4P/ D2 (4)

(dT/dt) = change in temperature over time (C s1),

Cp = specific heat of whey (3.9365 kJ kg1 C1),P =ultrasonic power (W),

m = sample mass (kg),

V = sample volume (mL)

D = probe diameter (0.7 cm).

Microbiological analysis

All microbiological analysis was performed inaccordance with Kochs pour plate method (Sch-

legel, 1992). Thereby, after each treatment se-rial dilutions of whey solution (0.9 % NaCl) wereformed using physiological solution in order toevaluate the total viable cells count, the viable countof coliforms and the viable cell count of yeasts andmoulds. As control samples, serial dilutions of un-treated fresh whey, pasteurized whey and ultrasoni-cated whey werecreated. Furthermore, samples forthe evaluation of the total viable cells count werepour-plated in Tryptic Glucose Agar (Biolife, Italy)and incubated at 30 C for 72 h. Coliform bacte-

ria samples were pour-plated in Endo Agar (Biolife,Italy), while dishes were incubated at 37 C for 48 hand bacteria loads were counted. For the enumera-tion of the viable yeasts and moulds count sampleswere pour-plated in Sabouraud-Maltose Agar (Bi-olife, Italy) and incubated at room temperature for72 h. All microbiological analyses were conducted ina triplicate for each experiment.

Sensory analysis

Samples of untreated, thermally treated, soni-cated and thermo-sonicated whey were collected insterile glass bowls, closed and stored in a cool envi-ronment for 24 hours. Before sensory analysis they

were adjusted to a room temperature, encoded andequal quantities of each sample were poured intotransparent cups. In a room designed according toISO8589:2007 samples were then presented simul-taneously to each of the five assessors trained to per-

form sensory evaluation of whey. Each sample wasevaluated for colour, appearance, flavour and odourwhere each attribute could have been rated withnotes from 1 to 5. The average note of each attributewas multiplied with a predetermined weighting fac-tor which was observed by the Delphi method. Inthis manner, scores for each evaluated attribute wereobserved. Bysummarizing scores of each attribute afinal score for each particular sample was obtained.The maximum score that one sample could obtainwas 20 (Molnar and rsi, 1982; Filajdi et al.,1988).

Results

Whey composition

The average composition of whey is given inTable 1. According to data available from literature(Jelen, 2003; Jelii et al., 2008), all determinedparameters are in range characteristic for rennetcheese whey.

Microbial inactivation

Changes in total viable cells count

Figures 1a-c demonstrate the reduction of thetotal viable cell count obtained by ultrasonicationand thermo-sonication treatments in comparisonwith the reduction observed by simulation of con-ventional pasteurization processes.

In general, ultrasonication treatments showed

no impact on the reduction of the total viable cellscount regardless the applied amplitude of ultrasonicwave and exposure time. Some treatments, especial-ly those of applying power level of 80 %(Figure 1b)

Table 1. Average composition of fresh rennet whey

n=5

pH SH Lactose (%)Total proteinamount (%)

Ash (%) Total solids (%) Milk fat (%)

6.160.22 5.920.30 4.890.06 1.710.20 0.570.01 5.140.05 0.170.05

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Figure 1. Reduction of total viable cells count by ultrasonicationat a) 400 W (power level 100 %), b) 320 W (powerlevel 80 %) and 240 W (power level 60 %) and bythermo-sonication with preheating to 55 C, 45 Cand 35 C during 5; 6.5 and 8 min of exposure in

comparison to pasteurized whey

and of power level 60 % (Figure 1c), re-sulted even in a slight increase of the to-tal viable cells count. This phenomenonmight be the consequence of shatteringthe clusters of microbial cells without

having a lethal effect at the same time.All applied thermo-sonication treat-ments resulted in the reduction of thetotal viable cells count where the ob-served reduction increased with expo-sure times and preheating temperatures.

As shown in Figure 1a, thermo-sonication treatments at power level 100% (400 W) combined with preheatingto 45 C and 55 C as well as exposure

times of 6.5 min and 8 minresulted in avery good reduction of the total bacte-ria number.Thereby, thermo-sonicationtreatment denoted as 100 %-55 C/8resulted in a greater reduction of the to-tal viable cells count (2.46 log cycles) incomparison with that observed by simu-lation of both pasteurization processes.Thermo-sonication treatment denotedas 100 %-55 C/6.5 resulted in betterreduction (1.94 log cycles) than simu-

lated pasteurization process at 65 for30 min (1.81 log cycles). The ultrasonicintensity of these treatments was calcu-lated to be 64.42 W/cm2 and 65.60 W/cm2 which is much lower in comparisonwith intensities calculated for sole ultra-sonication treatments for the same ex-posure times (Table 2b). However, datacontained in Table 2amight provide anexplanation for such results since dur-

ing thermo-sonication treatments de-noted as 100 %-55 C/8 and 100 %-55C/6.5 temperatures of approximately70 C are developed. Previously it hasbeen suggested that redundant increaseof temperature could minimise the ef-ficiency of killing bacteria by ultrasonicinduced cavitation (Villamiel and deJong, 2000). Raso et al. (1998) alsofound that at temperatures above 58 Cthe inactivating effect is solely due to

the heat. According to that and to thefact that whey samples were exposed

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Table 2a. Change in temperature of whey samples during ultrasonication and termo-sonication treatments

Sample Initial T (C) Final T (C) T (C)

Power level 100 % (400 W)

100 %/5 20.50.7 46.40.6 25.70.9100 %/6.5 20.60.7 50.60.7 30.00.9

100 %/8 20.80.4 59.70.6 38.90.9

100 %-55 C/5 54.90.1 67.60.3 12.70.3

100 %-55 C/6.5 54.90.2 690.2 14.10.1

100 %-55 C/8 54.90.1 70.80.3 15.80.2

100 %-45 C/5 450.2 61.90.2 16.90.4

100 %-45 C/6.5 450.2 65.10.3 20.10.5

100 %-45 C/8 450.1 66.60.2 21.60.2

100 %-35 C/5 350.2 54.50.6 19.50.7100 %-35 C/6.5 350.2 58.70.4 23.60.6

100 %-35 C/8 350.1 62.80.2 27.80.2


80 %/5 20.20.8 38.50.6 18.11.0

80 %/6.5 20.60.9 43.10.3 22.50.5

80 %/8 20.80.6 52.60.5 31.80.9

80 %-55 C/5 550.1 62.50.8 7.40.8

80 %-55 C/6.5 550.1 63.80.7 8.70.7

80 %-55 C/8 55.10.2 660.3 10.90.280 %-45 C/5 450.1 57.90.3 130.4

80 %-45 C/6.5 450.1 60%.70.3 15.70.4

80 %-45 C /8 450.1 620.2 170.1

80 %-35C/5 350.2 50.20.3 15.20.4

80 %-35 C/6.5 350.2 53.10.3 18.20.3

80 %-35 C/8 350.1 55.40.4 20.50.4


60 %/5 20.40.6 32.50.6 12.30.3

60 %/6.5 20.50.5 36.50.9 16.01.460 %/8 20.40.5 43.10.8 22.70.8

60 %-55 C/5 550.1 61.60.7 6.50.6

60 %-55 C/6.5 550.1 62.80.7 7.70.8

60 %-55 C/8 55.10.1 62.30.6 7.20.6

60 %-45 C/5 450.2 520.7 7.10.6

60 %-45 C/6.5 450.2 53.60.4 8.60.4

60 %-45 C/8 450.1 56.10.1 110.1

60 %-35 C/5 350.0 45.60.4 10.60.4

60 %-35 C/6.5 350.0 47.40.3 12.40.360 %-35 C/8 34.90.1 49.80.2 14.90.3

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to temperatures above 70 C for approximately oneminute, the observed reduction of the total viablecells count at this specific treatments is due to heatrather than to ultrasound induced cavitation.

Thermo-sonication treatments at power level80 % (320 W) combined with preheating to 55C and exposure times of 6.5 and 8 min resultedin the same or better reduction in comparison withsimulated pasteurization batch process at 65 C for30 min (Figure 1b). According to data presentedin Table 2a, the temperature of those samples in-creased up to approximately 66 C. The calculatedultrasonic intensities remained half lower than incase of sole ultrasonication treatments at the same

exposure times (Table 2b). Therefore the obtainedresults were probably due to the heat development

during thermo-sonication treatment as Raso et al.(1998) previously suggested.

Moreover, it can be observed that reductionobtained by treatments with preheating to 35 Cand exposure times of 6.5 min and 8min exceededreduction obtained in treatments with preheatingto 45 C and same exposure times. Data containedin Table 2a address how temperature increase wassomewhat higher during thermo-sonication treat-ments with preheating to 35 C. That might haveimproved the bactericidal effect of ultrasonicinduced cavitation. Additionally, this presumptionis supported by comparison of the calculated ultra-sonic intensities which are much lower in the case of

thermo-sonication treatments combined with pre-heating to 45 C as shown in Table 2b.

Table 2b. Calculated acoustic energy densities (AED) and ultrasound intensities (UI) for selected thermo-sonication treatments which resulted in greater microbial inactivation than simulated conven-tional pasteurization processes

TreatmentAED

(W/mL)UI (W/cm2)

Microbial reduction better

than pasteurization process at72 C/20 sec

Microbial reduction better

than pasteurization process at65 C/30 min

Totalviable

cells

Coliformbacteria

Yeastsand

moulds

Totalviable

cells

Coliformbacteria

Yeastsand

moulds


100 %-55 C/8 0.250.03 65.60%7.43 + + + + + +

100 %-55 C/6.5 0.250.01 64.422.22 - + + + + +

100 %-55 C/5 0.830.00 59.850.83 - + - - + +

100 %-45 C/8 0.330.01 84.743.13 - - + - - +

100 %-45 C/6.5 0.310.01 79.371.71 - - - - - +


80 %-55 C/8 0.140.01 35.632.29 - + + + + +

80 %-55 C/6.5 0.130.01 34.652.30 - + + + + +

80 %-45 C/8 0.230.01 58.913.62 - + + - + +

80 %-45 C/6.5 0.210.01 54.191.20 - + + - + +

80 %-35 C/8 0.270.01 70.401.00 - + + - + +

80 %-35 C/6.5 0.270.01 70.401.00 - - + - - +


60 %-55 C/8 0.080.01 21.533.20 - - + - + +60 %-55 C/6.5 0.090.00 23.470.88 - + + - + +

60%-55C/5 0.080.01 20.352.51 - - + - + +

60 %-45 C/8 0.130.01 33.912.23 - + - - + -

60 %-45 C/6.5 0.100.01 26.071.37 - + - - + -

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As shown in Figure 1c, neitherof the thermo-sonication treat-ments at power level 60 % (240W) resulted in better reduction ofthe total viable cells count in com-

parison with simulated pasteuriza-tion processes. However reduc-tion obtained by treatments withpreheating to 55 C and exposuretimes of 6.5 min and 8 min reachedvery similar levels (approximately1.7 log cycles) as simulated pas-teurization batch process at 65 Cfor 30 min (1.8 log cycles). It is no-table that the temperature increaseof these samples was not exces-sive (Table 2a), but the calculatedultrasonic intensities were morethan half lower than in the case ofultrasonication treatments at thesame exposure times. Consequent-ly, heat pre-treatment probablycaused an improvement of antimi-crobial effect of ultrasonic inducedcavitation.

Changes in viable coliform bacteria

count

Figure 2a-c demonstrates re-duction of viable coliform bacteriacount obtained by ultrasonicationand thermo-sonication treatmentsin comparison with reduction ob-served by simulation of conven-tional pasteurization processes.

Likewise in the case of total

viable cell count reduction, ultra-sonication treatments showed noimpact on the reduction of the vi-able coliform cells count regardlessthe applied amplitude of ultrasonicwave and exposure time. Also,slight increase in viable cell count isevident, especially after ultrasoni-cation at power level 60 %. Accord-ing to data presented in Table 2a,

the final temperature of the ultra-sonicated samples ranged between

Figure 2. Reduction of viable coliform bacteria count by ultrasonica-tion at a) 400 W (power level 100 %), b) 320 W (powerlevel 80 %) and c) 240 W (power level 60 %) and by ther-mo-sonication with preheating to 55 C, 45 C and 35 C

during 5; 6.5 and 8 min of exposure in comparison to pas-teurized whey

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32.50.6 C (exposure time 5 min) and 43.10.8C (exposure time 8 min). Coliform bacteria belongto mesophillic bacteria and prefer growth temper-atures from 37 C to even 49 C (Fotadar et al.,2005). Thereat generation time can be as short as 20

minutes if the temperature is high enough (Smith,1985). In accordance to that, utrasonication itselfprovides convenient growth temperatures for colif-orm bacteria, especially at power level 60 %.

As presented in Figure 2a, the best reductionwas observed when applying power level 100 % andpreheating to 55 C. Thereby regardless the expo-sure times, the obtained reduction levels (1.20 to1.79 log cycles) were higher than in the case ofsimulated pasteurization processes (0.98 to 1.12 log

cycles). Heat development during these treatmentsresulted in the samples reaching a final temperatureup to 71 C, which implies that the inactivationeffect was probably due to the heat solely as Vil-la miel and de J ong (2000) previously suggested.

Regarding thermo-sonication treatments atpower level 80 %, reduction obtained after treat-ments with preheating to 45 C and 55 C and expo-sure times of 6.5 min and 8 min exceeded reductionobtained by simulation of both pasteurization proc-esses, as shown in Figure 2b. Similar results wereobserved in treatments with preheating to 35 Cand exposure times of 6.5 min and 8 min as wellas in treatment denoted as 80 %-45 C/5 wherethe obtained reduction reached the same levels assimulated batch pasteurization process (65 C for 30min). According to data in Table 2a, heat develop-ment during these treatments might be responsiblefor such inactivating effects, which is in concordancewith findings of Ra so et al. (1998). However, unlikeRaso et al. (1998), in the present study no addi-

tional hurdle such as pressure was applied. Addition-ally, from data presented in Table 2b, it is evidentthat the ultrasonic intensity of highlighted thermo-sonication treatments rises conversely to preheatingtemperature and final temperature of whey samples.In such a manner the obtained inactivating effect ismost probably due to synergistic acting of heat andultrasonic cavitation.

As presented in Figure 2c, all thermo-sonicationtreatments at power level 60 % in combination withpreheating to 45 C or 55 C resulted in better reduc-tion of viable coliform bacteria count in comparisonwith simulated batch pasteurization process (65 C

for 30 min). Thereat treatments denoted as 60 %-45C/6.5 and 60 %-45 C/8 even exceeded reductionlevels in comparison with simulated pasteurizationprocess at 72 C for 20 sec, although heat develop-ment did not result in final temperature increase

above 56.2 C (Table 2a). As previously discussed,the inactivating effect of thermo-sonication treat-ments at power level 60 % relays also most probablyon heat improved ultrasonic cavitation.

Changes in viable yeasts and moulds count

Figure 3a-c demonstrates reduction in the vi-able yeasts and moulds count obtained by ultrasoni-cation and thermo-sonication treatments in com-parison with reduction observed by simulation ofconventional pasteurization processes.

Neither ultrasonication treatment showed animpact on the reduction of the yeasts and mouldsviable count regardless the applied amplitude of ul-trasonic wave and exposure time.

As presented in Figure 3a, the best reductionwas observed when applying power level 100 % andpreheating to 45 C or 55 C. The obtained reduc-tion during these treatments exceeded effects ofsimulated pasteurization batch process (1.34 log cy-

cles) regardless exposure times. According to Table2a, heat development during thermo-sonication atpower level 100 % and preheating to 45 C or 55C treatments resulted in final temperature of thesamples ranging from 62 C to 71 C. This impliesthat the inactivation effect was probably due to heatsolely as Villamiel and de Jong (2000) and Rasoet al. (1998) previously suggested.

The increase of exposure time and preheatingtemperature has been accompanied with increase

in the reduction level regarding thermo-sonicationtreatments at power level 80 %, as shown in Figure3b. Furthermore, all thermo-sonication treatmentswith preheating to 55 C as well as those with pre-heating to 45 C or 35 C and exposure times of 6.5and 8 min resulted in better inactivation in compari-son with effects obtained by simulation of both pas-teurization processes. Relating to data in Tables 2a-band to findings of Raso et al. (1998), as previouslydiscussed in case of coliform bacteria reduction, theobtained effects relay on heat improved ultrasonic

cavitation.

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According to Figure 3c, ther-mo-sonication at power level 60 %resulted in the best reduction whenpreheating to 55 C was applied.Regardless exposure time, all men-

tioned treatments were more effec-tive than simulated pasteurizationprocesses.

Sensory evaluation

The results of the sensoryevaluation of whey samples afterthermo-sonication and ultrasonica-tion in comparison with fresh andpasteurized whey are shown in Fig-ures 4 a-c. In general, all sonicatedand thermo-sonicated whey sam-ples obtained higher average scoresthan fresh or pasteurized samples.Mouth feel of ultrasonicated andthermo-sonicated whey samplesultrasonication thermo-sonicationwas considerably better. There wasno occurrence of sediment in any ofthose samples and colour remained

unchanged in almost all samples.Reek-Jambrak et al. (2008)studied the influence of high in-tensity ultrasound on functionalproperties of whey proteins. Theobtained results showed that ultra-sonication significantly improvedthe solubility of all tested solu-tions. Additionally, the homogeni-zation effect of ultrasound is alsowell known (Reek-Jambrak et

al., 2009). These two phenomena -increase in whey protein solubilityand homogenization effect - mightbe responsible for the general im-provement of fresh whey sensoryproperties after ultrasonication andthermo-sonication.

Flavour was slightly sweeterwhile odours of cooked milk werenot noticed. However, samples

where power level of 100 % (400W) was applied had considerably

Figure 3. Reduction of viable yeasts and moulds count by ultrasonica-tion at a) 400 W (power level 100 %), b) 320 W (powerlevel 80 %) and c) 240 W (power level 60 %) and by ther-mo-sonication with preheating to 55 C, 45 C and 35 C

during 5; 6.5 and 8 minutes of exposure in comparison topasteurized whey

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brighter colour and a metallic aftertaste. This wasespecially intensive after each thermo-sonicationtreatment with preheating to 55 C. Furthermore,all samples that were treated with power level 100 %in combination with preheating to 35 C had poorer

mouth feel in comparison with samples which werepreheated to 45 C or to 55 C. Therefore, sampleswhere power level 100 % was applied after preheat-ing to 45 C received the highest scores in sensoryevaluation as shown in Figure 4a.

The same observations were noticed in sampleswhere power level 80 % was applied in combinationwith preheating to 35 C or 55 C. However, thesephenomena were less expressed why these samplesreceived higher scores of sensory evaluation than in

case above. As shown in Figure 4b, samples whichwere preheated to 45 C received also in this casethe highest scores in sensory evaluation.

As shown in Figure 4c, samples where powerlevel 60 % was applied solely or in combinationwith preheating to 55 C, received somewhat low-er scores of sensory evaluation in comparison withsamples which were preheated to 35 C or to 45C. Samples treated solely by ultrasound had poormouth feel and slightly expressed odour of cookedmilk, while samples which were preheated to 55 Chad a metallic odour and rubbery aroma which is incorrespondence with results obtained by Riener etal. (2009). They studied the influence of high in-tensity ultrasound on sensory properties of raw milkby determining volatile compounds developed inmilk during ultrasonication. Volatiles detected byGC spectrometry were predominantly hydrocar-bons and are probably of pyrolytic origin, possiblygenerated by high localized temperatures associatedwith cavitation phenomena. Some of the detected

volatiles are believed to cause the occurrence of therubbery aroma in milk, which was less intense byreduction of the sonication power from 400 W to100 W.

Relating to all above mentioned observations,regardless the ultrasound power input, preheating to55 C results in occurrence of a metallic aftertastein thermo-sonicated samples. Furthermore, preheat-ing to 35 C, as well as applying power level 60 %(240 W), resulted in somewhat poorer mouth feelin comparison to other thermo-sonicated and ultra-sonicated samples.

Figure 4. Sensory evaluation of whey samples af-ter ultrasonication at a) 400 W (powerlevel 100 %), b) 320 W (power level 80%) and c) 240 W and after thermo-son-ication with preheating to 55 C to 55C, 45 C and 35 C during 5, 6.5 and 8

minutes of exposure compared to freshand pasteurized whey

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Conclusions

In general, the investigated ultrasonicationtreatments showed no impact on microbial inactiva-tion regardless the ultrasound power input and ex-

posure times.Thermo-sonication treatments with pre-heating

to 55 C showed good impact on microbial inactiva-tion but that effect is probably due to heat devel-oped during the treatment.

Microbial inactivation caused by thermo-son-ication treatments with pre-heating to 35 C and45 C increased with power level and/or exposuretimes. Better effects than those obtained by simula-tion of conventional pasteurization processes are ob-

tained at longer exposure times (6.5 and 8 min) andregarding reduction of coliform bacteria and yeastsand moulds.

Regarding calculated ultrasonic intensities andtemperature increase of samples during these treat-ments, it can be concluded that the obtained micro-bial inactivation might be a result of heat supportedultrasonic cavitation. However, additional studieswhere same exposure times and power inputs wouldbe applied at constant preheating temperaturesshould be carried out in order to support this thesis.

Ultrasonication and thermo-sonication treat-ments considerably improved sensory properties ofrennet cheese whey in comparison with simulatedpasteurization processes.

Thermo-sonication treatments at ultrasoundpower inputs of 400 W and 320 W in combinationwith preheating to 45 C, regardless exposure time,appeared to be optimal for the purpose of the sen-sory properties of whey improvement.

Overall, it can be concluded that future stud-

ies should focus on processing parameters optimiza-tion at latter conditions in order to obtain adequatereduction of total bacteria count by applying longerexposure times and/or varying cycles.

Acknowledgements

This study was supported by the Croatian Min-istry of Science, Education and Technology (Project058-0000000-0443).

Utjecaj i usporedba toplinske obrade,ultrazvuka i termosonifikacije na

mikrobioloku kvalitetu i senzorskasvojstva slatke sirutke

Saetak

Ultrazvuk i termosonifikacija ubrajaju se u al-ternativne, netermalne metode procesiranja hrane.Stoga je svrha ovog rada bila ispitati utjecaj razliitihulaznih snaga ultrazvuka (240 W, 320 W, 400 W) sai bez primjene umjerenih temperatura na mikrobi-oloku kvalitetu i senzorska svojstva svjee slatke

sirutke te usporediti s utjecajem toplinske obradena iste parametre. Obrada ultrazvukom nije poka-zala nikakav utjecaj na redukciju bilo koje skupinepraenih mikroorganizama. Tretmani termosonifika-cije uz predgrijavanje uzoraka na 35 C, odnosno 45C rezultirali su mikrobiolokom redukcijom koja jerasla proporcionalno trajanju tretmana i/ili ulaznojsnazi, a vjerojatno je posljedica kavitacije pojaaneuinkom topline. Najuspjenijima su se pokazali tre-tmani termosonifikacije uz predgrijavanje uzoraka na55 C i primjenu ulazne snage 400 W te su rezultiraliveom mikrobiolokom redukcijom nego simuliranipostupci pasterizacije. Meutim, dobivena redukcijavjerojatno je posljedica djelovanja razvijene topline.Obrada ultrazvukom i termosonifikacijom rezultira-la je znaajnim poboljanjem senzorskih svojstava si-rutke u odnosu na simulirane postupke pasterizacije.Punoa okusa uzoraka sirutke bila je znaajno bolja,nije uoena pojava taloga, a boja je ostala nepromije-njena u gotovo svim uzorcima.

Kljune rijei: ultrazvuk, termosonifikacija,senzorska svojstva, mikrobioloka redukcija,slatka sirutka

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