Research articleInstitute of Brewing & Distilling

Received: 9 August 2013 Revised: 4 December 2013 Accepted: 5 December 2013 Published online in Wiley Online Library: 9 January 2014

(wileyonlinelibrary.com) DOI 10.1002/jib.113

Dissolved oxygen content in apple must:technological implications in cider processingAline Alberti, Cíntia Maia Braga, Henrique Jaster and Alessandro Nogueira*

Oxygen can influence the composition and quality of apple must and cider. In this study, the effect of unit operations on oxygendissolution during apple must processing was investigated and the technological implications discussed. Oxygen dissolutionafter different apple must processing unit operations, and also the effect of enzymatic browning reaction on dissolved oxygenuptake were analysed. During pressing, the highest and first oxygen dissolution occurred in the must at 6.8mg/L, on average.The enzymatic browning reaction after 10min was responsible for an oxygen uptake above 80%. The addition of sulphurdioxide or maceration of crushed apple results in amust withmore oxygen content, however the beverage colour is negativelyaffected. The incorporation of oxygen into themust by bubbling oxygen or themovement of liquidmay not be effective owingto consumption by the reaction of residual polyphenol oxidase. Moreover, this can affect the alcoholic fermentation becauseof the oxidation of nutrients such as vitamins. If the apple must is depectinized, racked and filtered, there will be enoughoxygen to obtain a quality cider. These results and observations can be useful for selecting unit operations in cider companiesin order to improve the beverage quality. Copyright © 2014 The Institute of Brewing & Distilling

Keywords: enzymatic browning; alcoholic fermentation; yeast; cider

* Correspondence to: Alessandro Nogueira, Food Science and TechnologyGraduate Programme, State University of Ponta Grossa, 4748 CarlosCavalcanti Av., Uvaranas Campus. CEP 84.030-900 Ponta Grossa, PR, Brazil.E-mail: alessandronog@yahoo.com.br

Food Science and Technology Graduate Programme, State University ofPonta Grossa, 4748 Carlos Cavalcanti Av., Uvaranas Campus. CEP 84.030-900Ponta Grossa, PR, Brazil 65

IntroductionCider is a sparkling apple wine that is produced in more than25 countries. It can be produced with dessert and/or cider applevarieties and its unit operations are similar, but may varybetween producers (1). However, in any kind of processing, ifthere is no antioxidant addition there will be competition foroxygen between the yeasts and enzymatic browning.

In cider processing, the unit operations such as pressing,centrifugation, racking and filtration are responsible for oxygendissolution in the apple must (1,2). However, when the fruit iscrushed and pressed, the contact between the phenolic com-pounds and polyphenol oxidase (PPO), in the presence of oxygen,triggers the enzymatic browning process (3–8). This reaction canreduce the dissolved oxygen content in the must, which can bedetrimental to yeast growth (Saccharomyces cerevisiae) (9)

In alcoholic fermentation, oxygen can be a limiting elementfor yeast growth and can affect aroma production, mainly thosearomas synthesized by apiculate yeasts (10–12). Under cider pro-cessing conditions, the yeast (Saccharomyces uvarum) needs2.0–2.5mg/L of dissolved oxygen (2). At the beginning of alco-holic fermentation, this element is used in different pathwayssuch as classical respiration, sterol biosynthesis, oxygen detoxifi-cation and with other oxidases. These compounds play animportant role in the permeability of the cell, affecting its viability(reproductive capacity) and vitality (the ability to produce energy)(12–14).

In cider and wine processing, the addition of nutrients (nitro-gen and vitamins), and shaking or moving the must to dissolvethe oxygen, are common practices used to prevent stuck or slug-gish fermentations. In addition, there are unit operations such ascrushed apple maceration and sulphiting, before or after theapple must extraction, which can influence the concentration ofdissolved oxygen. However, oxygen can positively or negativelyinfluence the composition and quality of the cider. This area of

J. Inst. Brew. 2014; 120: 65–70 Copyright © 2014 The Institu

the process has been poorly studied and therefore this workanalyses the oxygen dissolution after unit operations of extrac-tion and treatment of apple must and also discusses the techno-logical implications for the quality of the cider.

Materials and methods

Materials

Samples of five dessert apple varieties (Eva, Fuji, Gala, GrannySmith and Red Delicious), provided by the Boutin Fruit CultureCompany (Paraná, Brazil), were used in this study.

Methods

Effect of oxygen uptake by enzymatic browning reaction.Samples containing around 2.5 kg of five apple cultivars (Eva,Fuji, Gala, Granny Smith and Red Delicious) were used. The fruits,selected and washed, were milled in a microprocessor (MetvisaProcessor, Type MPA, number four chopping blade) and thecrushed apple was macerated for 0 (without maceration), 5, 10,20 and 30min at room temperature (22 °C), without agitation,in polypropylene containers (450 × 350 × 45mm). Then, thecrushed apple was conditioned and stacked in polyethylenescreen packages, submitted to a pressure of 3 kgf/cm2 for3min (Hydraulic Press Eureka, Hoppe Ind. Ltda, Brazil) and the

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A. Alberti et al.Institute of Brewing & Distilling

66

dissolved oxygen in the apple must was determined (oxygenmeter Digimed, DM-4, equipped with an electrode with a silveranode, platinum cathode and polytetrafluoroethylene mem-brane) and was monitored for 10min. The apple must wasdepectinized with enzyme (Pectinex 3XL, Novozymes) in theproportion of 3mL/hL (60min at 45 ºC), and after 100min atroom temperature (20–26 °C) it was racked (hose of 1m with0.5mm diameter) and filtered (qualitative filter paper, Qualy).At all steps of the processing of the apple must, the oxygencontent and temperature of the must were recorded. All of theexperiments were performed at 975m above sea level, withtemperatures between 20 and 26 °C, and at an atmosphericpressures of 1008–1015 hPa.

Quantification of the dissolved oxygen content in differentprocessing unit operations. Samples containing around5.0 kg of apple varieties (Gala and Fuji) were selected, washed,crushed (Metvisa Processor, Type MPA, number four chopping)and pressed (Hydraulic Press Eureka, Hoppe Ind. Ltda, Brazil).The dissolved oxygen content (Oxygen meter Digimed, DM-4)and temperature were analysed in the apple must at pressing(pressing and a fall 0.40 m until the beaker) and in the following10min, and in the apple must after the clarification treatments.An amount of 3.0mL/hL of pectinase (Pectinex, Novozymes)was added to the apple must (40 °C), and after 100min at roomtemperature (20–26 ºC) the supernatant was racked and the oxy-gen content and temperature were determined. To evaluate theeffect of sulphur dioxide on the dissolved oxygen content duringunit operations, potassium metabisulphite was used. It wasadded immediately after crushing at concentrations of 50, 100and 150mg K2S2O5/kg of apples.

Statistical analysis. Variances results were analysed using theF-test (two groups) or by Hartley’s test (p> 0.05). Differencesamong the groups were assessed by means of the Student’s t-testfor independent samples (two groups) or one-factor ANOVAfollowed by Tukey’s test. A p-value below 0.05 was consideredsignificant. Statistical analyses were performed using Statistica7.0 (StatSoft Inc., USA) and Microsoft Office Excel 2010®.

Results and discussion

Effect of oxygen uptake on enzymatic browning reactions

The average content of dissolved oxygen in the apple must atthe pressing time in the five apple varieties that were usedwas 6.8 ± 0.4mg/L (Fig. 1). However, the oxygen incorporatedin the pressing was quickly used by the PPO in the enzymatic

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Time, min

(a)

Figure 1. Oxygen uptake (mg/L) by the enzymatic browning reaction in apple must in thEva (♦), Fuji (□), Gala (▲), Granny Smith (Δ) and Red Delicious (■); and (b) Gala apple mu

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browning reactions (15,16). The apple must of the Fuji, Red Deli-cious and Gala varieties had a higher biochemical consumptionof dissolved oxygen. In 3min, the decrease was 97.8, 96.9 and93.1%, respectively, and it remained constant during 10min ofanalysis. In the apple musts with lower biochemical oxygenutilization, Eva (82.8%) and Granny Smith (86.1%), the uptakerate by the enzymatic browning reaction was lower (Fig. 1a).The amount of oxygen used in the enzymatic browning reactionis related to the composition of the apple must (17), mainlyowing to the phenolic profile of the variety. The polyphenoloxidase reacts preferentially with chlorogenic acid, p-coumaricacid and catechin (18–21).

The maceration of crushed apple resulted in a smaller reduc-tion in dissolved oxygen uptake of the apple must by enzymaticbrowning reactions (Fig. 1b). The average dissolved oxygen, im-mediately after pressing (7.4 ± 0.5mg/L), was similar to the applemust without the maceration treatment (6.8 ± 0.4mg/L). How-ever, after 10min, oxygen levels were significantly (p< 0.05)higher than the treatment without maceration, reaching levels10 times higher in the treatment with 5 and 10min of macera-tion (Fig..1b). The operation of maceration caused the oxidationof phenols before pressing. Thus, the enzyme used less oxygen,incorporated in the pressing in the oxidation reactions, sincethe content of phenolic compounds (substrates) which can beoxidized by the enzyme was lower.

Effect of dissolved oxygen content during unit operations

The evolution of oxygen during the different unit operations ofapple must, with and without maceration treatment, can beseen in Table 1. The dissolved oxygen content after filtrationshowed no significant difference (p> 0.05) between both treat-ments. However, if this operation was not performed, thedissolved oxygen in the must without maceration would be<2.0mg/L. According to Nogueira et al. (2), the content requiredfor complete fermentation in French apple musts is 2.5mg/L.Thus, the apple must fermentation without maceration treat-ment might not have enough oxygen for a complete alcoholicfermentation or may have deficiencies in aroma synthesis.

Table 2 shows the effect of different maceration times on thedissolved oxygen. After pressing, the oxygen content in theapple must in all of the treatments was similar. After 10min,the oxygen reduction was greater in the treatment without mac-eration (96.9%). In the maceration times of 5, 10, 20 and 30min,the reductions ranged from 49.3 to 66.2%, after 10min of press-ing. The largest reduction, which corresponds to the must beingmacerated for 30min, may be due to the action of endogenous

0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0

Time, min

(b)

e first 10min after pressing operation: (a) without maceration in apple must varieties –st with different maceration times – 5min (♦); 10min (▲); 20min (○) and 30min (■).

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Table 1. Evolution of dissolved oxygen during Gala apple must processing in the presence (n= 6) and absence (n= 8) of amaceration operation

Unit Operation

Oxygen dissolved, mg/L

p (F-test) p (Student t-test)Maceration No maceration

Pressing (instantaneous) 7.4 ± 0.5 6.8 ± 0.4 0.71 0.11(after 10min) 3.2 ± 0.2 0.3 ± 0.2 0.94 <0.001

Pectinase addition and homogenizationb 4.3 ± 0.3 2.6 ± 0.3 0.91 <0.001Heating at 40°Cc 1.3 ± 0.3 0.4 ± 0.2 0.25 <0.01End of depectinization processd 1.8 ± 0.2 1.4 ± 0.3 0.91 0.05Rackinge 2.8 ± 0.2 1.8 ± 0.3 0.99 <0.01Filtered mustf 3.4 ± 0.2 3.0 ± 0.3 0.65 0.08

Note: n= replicate number; a20min of maceration; b13 min after pressing; cfor enzyme action, 20min after pressing; d120min afterpressing; e125 min after pressing; f150 min after pressing.

Table 2. Effect of different maceration times (0, 5, 10, 20 and 30min) on the dissolved oxygen content (mg/L) during Gala applemust processing

Macerationtime (min)

Pressing

Racking musta Filtered mustbInstantaneous After 10min

O2 Temperature (ºC) O2 Temperature (ºC) O2 Temperature (ºC) O2 Temperature (ºC)

0 6.5CDa 24.4 0.2 Dc 25.5 1.8 Cb 28.3 2.9 Cb 28.15 7.6 Aa 25.0 3.7 Ab 26.0 3.7 Ab 29.0 3.9 Ab 28.810 8.0 Aa 25.1 3.0 Bb 25.8 2.4 Bbc 28.5 3.4 ABb 28.020 7.3 ABa 24.8 3.4 Ab 25.5 2.7 Bc 28.8 3.5 Ab 28.430 6.8 BCa 25.3 2.3 Cb 26.4 2.6 Bb 29.0 2.8 Cb 28.7

Note:a130 min after pressing; b150 min after pressing. Different capital letters indicate significant differences (p< 0.05) betweencolumns; different small letters indicate significant differences (p< 0.05) between lines.

Dissolved oxygen content in apple mustInstitute of Brewing & Distilling

enzymes of the fruit that caused the release of phenols, which innormal processing (not maceration) would be retained in thepomace (5,22). With higher levels of phenols, the enzyme hasmore substrate for the enzymatic oxidation reaction, which canthus continue. Therefore, maceration for 20min is sufficient tomaintain a high level of oxygen in the apple must. After theracking and filtration operations, the dissolved oxygen was keptabove 2.4mg/L in all of the maceration times (Table 2).

67

Effect of sulphur dioxide on dissolved oxygen content duringmust processing

Sulphur dioxide is widely used in oenology and cider processing.When it is added to apple must, it inhibits or kills most spoilageyeasts, moulds and bacteria, while enabling the growth of desir-able fermenting yeasts (such as Saccharomyces cerevisiae orS. uvarum) (23). Therefore, the sulphite reduces browning byinhibiting the action of PPO (24) when it is added to the crushedapple, a common practice in certain countries (1). Table 3 showsthat the dissolved oxygen remained around 5.55mg/L in two ap-ple must varieties, with three concentrations of sulphur dioxideadded to the crushed apple (50, 100 and 150mg/kg). However,the inhibiting mechanisms, in relation to enzymatic browning,are still not fully understood. Some authors have suggested that

J. Inst. Brew. 2014; 120: 65–70 Copyright © 2014 The Institu

competitive inhibition occurs by a binding to a sulphydryl groupat the active site of the enzyme (25,26).

Technological implications

The dissolved oxygen values presented in this study wereobtained in a small pilot plant laboratory. Larger equipmentand varying temperatures (higher concentrations dissolving atlower temperatures ≤5 °C) can influence the dissolution of oxy-gen, but the values would still be close to those obtained in thiswork. Ribéreau-Gayon et al. (23) and Du Toit et al. (27) evaluatedthe dissolution of oxygen in different unit operations during anindustrial processing of wine, which presented values between6 and 8mg/L with pumping-over, transfer from tank to tank(up to 6mg/L), filtration (4–7mg/L), racking (3–5mg/L), centrifu-gation (up to 8mg/L) and bottling (0.5–3mg/L). These values aresimilar to those found in the apple must in the present study(Fig. 1a). This similarity can be explained by the capacity of oxy-gen dissolution, which is dependent on temperature and on thecomposition of the liquid (13).It was observed that the reaction rate of the enzymatic brow-

ning of the apple must was rapid and was related to the applevariety (Fig. 1a). This reaction rate was obtained at 22 °C. It maybe slower at lower temperatures, although the kinetics of thisreaction are temperature-independent (27). This reaction can

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Table

3.Effect

ofsulphu

rdioxidead

ditio

non

dissolvedoxyg

en(m

g/L)

durin

gap

plemustprocessing

Cv.

Unitop

eration

Potassium

metab

isulph

iteconten

t(m

g/kg

)

050

100

150

O2

Tempe

rature

(ºC)

O2

Tempe

rature

(ºC)

O2

Tempe

rature

(ºC)

O2

Tempe

rature

(ºC)

Gala

Pressing

5.20

Aa

20.8

5.65

Aa

20.9

5.45

Aa

19.8

5.75

Aa

21.7

10min

afterpressing

0.45

BCb

19.7

5.50

Aa

19.9

4.95

Aa

19.4

5.80

Aa

19.3

Dep

ectin

ized

musta

1.70

BCb

22.5

5.05

Aa

22.3

5.25

Aa

23.5

5.30

Aa

22.8

Rackingb

2.10

ABb

22.0

5.35

Aa

24.1

5.75

Aa

23.5

5.65

Aa

23.1

Filte

redmustc

3.90

Ab

22.6

6.10

Aa

21.8

6.05

Aa

22.5

6.15

Aa

20.6

Fuji

Pressing

5.30

Aa

22.2

5.45

Aa

21.7

5.40

Aa

20.3

5.20

Aba

20.5

10min

afterpressing

0.00

Eb19

.95.20

Aa

20.7

4.35

Aa

19.2

5.35

Aa

19.6

Dep

ectin

ized

musta

1.70

CDb

22.6

4.85

Aa

23.6

4.45

Aa

22.8

4.60

BCa

22.9

Rackingb

2.20

Cb

22.8

4.85

Aa

23.6

5.10

Aa

22.9

5.00

Aba

23.0

Filte

redmustc

3.20

Bb

22.5

5.55

Aa

22.3

5.55

Aa

21.6

5.80

Aa

21.9

Note:Cv.,app

levarie

ty;a12

0min

afterp

ressing;

b13

0min

afterp

ressing;

c 150

min

afterp

ressing.Differen

tcapitallettersindicate

sign

ificant

differen

ces(p≤0.05

)betweencolumns,

differen

tsm

allletters

indicate

sign

ificant

differen

ces(p≤0.05

)be

tweenlin

es.

A. Alberti et al.Institute of Brewing & Distilling

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68

reduce around 70% of the total phenols and up 50% of the an-tioxidant potential in the apple must (4). The process of macera-tion of crushed apple, without the addition of enzymes,promotes a prior oxidation reaction. After pressing, the PPO issaturated or there is little phenolic substrate, so the remainingoxygen in the must after pressing becomes evident (Fig. 1b). Thisoperation tends to increase the concentration of melanoidins inthe must and consequently the cider will have a darker colour(28,29). Furthermore, Vidrih and Hribar (30) reported that themaceration process (24 h) caused an increase in iso-amyl alcohol,n-propanol, 2-phenyl ethanol, n-butanol, iso-butanol and metha-nol in ciders made from the Gala and Gloster varieties. However,this increase in higher alcohols also produced a negative correla-tion in terms of sensory evaluation.

In some countries (Argentina and Brazil) sulphur dioxide(100–200mg/L) is added to the crushed apple in order to blockthe enzymatic browning reaction and to provide a beverage witha lighter colour (1). The addition of sulphite to the crushed applesinhibits the PPO and consequently increases the dissolved oxy-gen content in the must (Table 3). The sulphur dioxide preventsoxidation because it binds to the enzyme, irreversibly inactivatingit, and owing to its reducing power it reduces the o-quinone pro-duced by PPO catalysis to the less reactive diphenol, interruptingthe reaction of the formation of melanoidins (16,25,26). In addi-tion, this compound has antimicrobial and bacteriostatic action,without affecting the Saccharomyces sp. yeast, which is resistantto the compound at a concentration that allows its use, eliminat-ing the competition between the yeast and other microorgan-isms (23). These two factors provided by the sulphite, (thehigher concentration of dissolved oxygen and the eliminationof competition of microorganisms with Saccharomyces sp.) com-bined with high temperatures (>25 ºC), make the alcoholic fer-mentation rapid, and it can be completed (exhaustion offermentative sugars) within 5 days (1). The sulphite (> 100mg/L)and rapid fermentation affect the formation of aromatic com-pounds, such as the major ethyl esters and higher alcohol acetates,which are favoured by slow fermentation (31,32), and induce therelease of acetaldehyde by Saccharomyces sp, which leaves theproduct with unwanted sensory notes of ‘aroma of yeast, neutral,less typically green, grassy and apple-like aroma’ (24,33)

The kinetics of the fermentation of apple must, obtained withor without maceration, with dissolved oxygen levels above2.5mg/L did not differ significantly (data not shown). Thesevalues were obtained after filtration of the apple must, but thereare producers who use only one racking after depectinizationand those who do not rack at all. In these cases, the amount ofdissolved oxygen would be <2.0mg/L (Table 1), which couldcompromise the kinetics of the fermentation by limiting thegrowth of yeasts, which could result in extra hours or days untilthe complete exhaustion of fermentable sugars. In some cases,owing to the lack of oxygen and the elimination of phytosterolsin the must (which may be used by the yeast in the biosynthesisof the sterols of the plasma membrane) (34), through amicrofiltration operation, there may be a halt in fermentation (35).

In order to avoid a stuck fermentation, owing to the lack of ox-ygen or nutrients, some companies that process wine and cideradd nitrogen sources to the must and perform homogenizationwith two goals: to dissolve the nutrients and to incorporate oxy-gen into the must (36). However, the incorporation of oxygeninto the must, either by bubbling oxygen or by the movementof liquid, may not be effective owing to consumption by thereaction of residual PPO. Moreover must oxygenation can affect

J. Inst. Brew. 2014; 120: 65–70te of Brewing & Distilling

Dissolved oxygen content in apple mustInstitute of Brewing & Distilling

the alcoholic fermentation because of the oxidation of nutrientssuch as thiamine, biotin and pantothenic acid, which are essentialvitamins for yeast growth (37–39). This negative effect of oxygencan result in up to twice the normal time for the complete utiliza-tion of fermentable sugars (data not shown). On the other hand, ifthis operation is performed using micro-oxygenation at the end ofthe cell growth phase, it will promote new cell multiplication oncethe oxygen has been utilized quickly by the yeasts (13,14).

The lack of oxygen at the beginning of fermentation can affectthe metabolism of apiculate yeasts that require oxygen for theformation of sensory aromas with ‘fruity’ and/or ‘floral’ notes, suchas ethyl ethanoate, ethyl octanoate, 3-methylbutyl ethanoate,3-methyl-1-butanol and ethanol, mixed with ethyl decanoate,hexyl ethanoate and 2 phenyletylacetate (10,40), which are rec-ognized as standards for the identity and quality of cider in somecountries. Some non-conventional yeast strains are sensitive tosulphite and others show some resistance (41); however, the for-mation of aromas, even with a viable strain can be affected (33).

6

ConclusionThe dissolved oxygen in apple must can influence the composi-tion and quality of cider. It is important for complete alcoholicfermentation and the aromatic quality of the cider. The pressingoperation incorporates the highest concentration of oxygen inthe apple musts, but the enzymatic browning reaction can con-sume it in <10min The addition of oxygen by bubbling, ormovement of the apple must, should be avoided owing to theoxidation of important nutrients. The effect will be positive onlywhen added in specific ways at the end of the growth phase.One way to minimize this consumption is by macerating thecrushed apple, but the cider will have a darker colour. The sul-phur dioxide, added to the crushed apple, inhibits the PPO,maintaining a level of dissolved oxygen that is higher thanneeded for the yeast, but the cider is lighter in colour and itcan lose aromatic quality. The more unit operations that areperformed, the greater the dissolution of oxygen; if the applemust is depectinized, racked and filtered, there should beenough oxygen to obtain a quality cider.

Acknowledgements

The authors are deeply grateful to Agrícola Boutin for the applesamples and the National Counsel of Technological and Scien-tific Development, Araucaria Foundation and Coordination forthe Improvement of Personnel in Higher Level for the financialsupport and scholarships offered during the implementationperiod of this research.

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