Journal of Scientific & Industrial Research VoJ.59, May 2000, pp 389-394

Inhibition of Enzymatic Browning in Apples, Potatoes and Mushrooms

Charanjit Kauri and Harish C Kapoor2* IDivision of Fruits and Horticultural Technology

2Division of Biochemistry Indian Agricultural Research Institute, New Delhi 110012, India

Received 29: November 1999; accepted: 0 I February 2000

Effect of different browning inhibitors like ascorbic acid (AA), 4-hexylresorcinol (HR) and banana leaf extract (BLE) either alone or in different combinations was studied on apples, mushrooms, and potatoes. The results were compared with the samples treated with sulphites which are banned because of their toxic effects. A mixture of HR. AA and BLE significantly inhibited the enzymatic browning during storage at 4"C. Similar decrease in polyphenol oxidase activity was also observed. The colour and texture of treated samples were closer to the fresh samples. Total viable microbial, yeast, and mould counts were also reduced. Overall performance of this formulation was found to be better than that of sulphites.

Introduction

The undesirable melanosis (browning) of fruits and vegetables is of great concern during post harvest han­dling and processing. Loss in colour often renders the product unacceptable by the consumer. Polyphenol oxi­dase (PPO, E.C.1.14. IS.I) which cause the oxidation of phenolic compounds is considered responsible for browning of many fruits and vegetables l

-,.

Sulphites have been extensively used as effective antibrowning agents in food industry but in recent years concern over their adverse health effects has resulted in severe limitations in the use of sulphite-based agents in the processed products('-x . This has stimulated research in finding alternative suitable antibrowning agentsY- 16•

Various anti-browning agents in use are ethanol, L­cystein, ascorbic acid, and its derivatives and kojic acid, etc. Recently the use of hexylresorcinol (HR) as an anti­browning agent in the treatment of apple slices 17 has been shown to reduce the browning in minimally processed products.

Banana leaves have been traditionally used in south India, for packaging of fresh food and also as serving plates for eatables. Although no scientific basi s on the possible preservative role of banana leaves is available, yet it is speculated that banana leaf extract may serve as a good source of natural preservative for fruits and veg-

* Author for correspondence

etables . Therefore the objective of the present study was to evaluate the performance of banana leaf extract (BLE) and HR, when applied individually or in combination with ascorbic acid as possible browning inhibitors and preservatives for apples, mushrooms, and potatoes_

Materials and Methods

Sample Preparations

Ripe apples (Royal delicious) and potatoes were obtained from local market. Freshly harvested mush­rooms were obtained from a local farm . Samples with no external defects were selected for treatment. Apples were cut into round pieces (0.5-0.7 cm); mushrooms were trimmed of their stems within 1-2 cm of the pileus (cap) and cut into two halves. Potatoes were also cut into 0.5-0.7 cm round pieces. Inbetween sampling the knife blade was immersed in 0.0 I per cent sodium hypochlorite so­lution so as to avoid bacterial contamination. Different treatments (Table I) were applied as dips in aqueous solutions of various browning inhibitors. Treated samples were packed in polythene bags, sealed, and stored at 01JC.

Preparation of Banana Leaf Extract (BLE) Banana leaves (500g) were cut into I cm long

pieces, washed thoroughly in running water, soaked in hot water (60°C) for 6h and filtered. Brown coloured extract obtained was decolorised by passing through activated charcoal and used as BLE.

390 J SCIIND RES VOL 59 MAY 2000

Table I - Composition of different treatments

Components Treatment solutions (ml)

T-I T-2 T-3 T-4 T-5 T-6 T-7 T-8 T-9 T- IO

Water 100 99.89 99.40 99.40 99.39 99.39 99.70

4-Hexyl resorcinol (HR)

Citric acid 0.50

Ascorbic acid (AA) 0.50

Sulphitcs

Bananna leaf extract (BLE) Calcium chloride 0.10 0.10 0.10

Enzyme Extraction

All the samples were frozen in liquid nitrogen. Fro­zen ti ssue were blended in 10 mM sodium phosphate buffer (pH 6.0) for I min and then centrifuged at 13,000 g for 10min. The supernatant was used as crude extract for enzyme (PPO) assay.

Enzyme Assay

Change in absorbance was followed at 420nm in Spectronic-2 J Spectrophotometer at lOs intervals I X. The assay mi xture contained 1.5 ml of standard phosphate buffer (pH7 .0), ] .5 ml of 0.02M catechol 11 fin a l conc. IO·2 M) and 10-15111 of crude extract so as to ob­tain a linear slope. Rate of retention was determined from the initial linear slope of the curve. The unit of enzyme acti vity was defined as the rate of change of one unit absorbance/m i n.

Assay of Browning Browning was measured as described by Coseteng

and Lee l9 Frozen sample (20g) was homogeni zed in 40 ml dist illed water for 2 min ancl centrifuged at 800 g for] 0 min in cold . One hour after homogenizat ion , 15 ml of 95 per cent ethanol was added to 10 ml of superna­tant , shaken, and centrifuged again at 800g for 15 min . Browning intensity was determined spectrophotometri­cally by tak ing absorbance at 440nm.

Microbial Analysis

Microbia l g rowth was evaluated aseptica ll y by homogenizing (25g) sample in 225 m] of 0. 15 peptone water and serially diluted. Each dilution (l ml) was plated into potato dex trose agar (PDA) and ac idified PDA for culture of bacteria, yeast, and moulds respective ly2o.21 . Yeast and moulds pl ates were cultured at 220C for 7 d.

0.01 0.01 0.0 1 0.01

0.50

0.50 0.50

0.20

99.90 99.89 99.39

0.10 0.10 0.10 0.10 0.10 0.10

Viable counts were determined by counting the number of colonies formed and reported as colony forming un its per gram sample (c.f.l1g)

Residual Analysis of HR

HR was extracted with ethyl acetate, purified and analyzed with high performance liquid chromatography (HPLC),' according to method described by King et ap2 . 20 III of HR sample preparation was injec ted into a 3.9x300 mm ] 0 mlm Bond C 18 column and eluted with gradient solvents of 0.1 per cent trifluoroaceti c acid (TFA) in water and 0.1 per cent TFA in acetonitrile at a flow rate of 1 ml/min. The column was flu shed with 100 perc ent methanol and re-equilibrated before the next injection . HR in the elute was detected by an UV detec­tor at 214 nm and quantified based on sample peak area as compared to standards.

Results and Discussion

Significant formation of brown pigments was ob­served on the surface of cut apples, mushrooms, and potatoes which was refl ected by significant increase in absorbance at 440 nm in control (untreated) sampl es, T-I (Table 2) . Browning reaction was reduced to a good extent by sulphite (T-7) and 0.0 I per cent HR (T-2 ) treat­ments. A combination of HR and ascorbic acid (T -6) was, however more effective in reducing the browning intensi ty. Dipping of samples in BLE (T-8) also inhib­ited browning to some extent in all the samples.However, supplementation of BLE with HR (T-9 ) and BLE with HR and AA (T-I 0) significantly inhibited the browning reaction and browning intensity was reduced by 60-65 per cent. Citric ac id a lone (T-3) or in combination with HR (T-5 ) was not so effec tive in inhibiting browning. A

KAUR & KAPOOR: ENZYMATIC BROWN ING 391

Table 2 - Effect of different treatments on the browning intensity after storage at o"t for four weeks

Treatments Browning intensity ( Absorbance at 440 nm)

Apples

T-I 0 .140i{1.009 (100)

T-2 0 .085±o.002 (60)

T-3 0.128± 0.009 (95)

T-4 0 .1 14±0.01 I (80)

T-5 0.085± 0.007 (62)

T-6 0 .075± 0.007 (55)

T-7 0 .086±o.016 (6 1)

T-8 0 .118± 0.009 (85)

T-9 0 .071± O.OOT (5 1)

T- IO 0 .05o± 0.005 (36)

CD 5 per cent 0.038

Values in parenthes is are percentage,Values are mean ± SEm

bleaching effect was observed in sulphite treated (T-7) samples whi le all the other treatments retained the fresh colors. Treatments T-8, T-9, and T- IO were most effec­ti ve in maintaining the lightness of samples .

The rate of browning during storage was influenced by the length of immersion of samples in the treatment solutions. Duraion of 5-7 min was found to be optimum for decreasing the browning reaction . Longer duration immersion may add to the diffusion of the compounds into the sample ti ssues.

Activity of PPO was moni tored in the frozen apples, mushrooms, and potatoes to observe re lati onship with degree of browning and PPO. A positi ve correlation be­tween degree of browning and PPO acti vi ty was observed in all the treatments. As ev ident from Table 3, treatment T- JO was again found to be the most effecti ve in reduc­ing the PPO acti vity (35-40 per cent). Combination of HR and AA (T-6) cou ld also reduce the enzyme acti vity and also enzymatic browning to the levels comparable to sulphites. Although the treatments like T-3 and T-4 were able to restrict browning to a certain extent by re-

Musrooms Potatoes

0 .2 10±0.005 (100) O.I±o.OO9 (100)

0 .1 24±o.012 (58) 0 .06±o.OO9 (55)

0 .200±0.009 (96) 0 . lo±o.OO5 (94)

0 .163± 0.009 (78) 0 .08±o.OO9(75)

0. 115±o.007 (56) 0 .06±o.OO5 (54)

0 .109±o.005 (52) 0 .05±o.OO5 (50)

0 .1 27±o.005 (60) 0 .05±o.OO5 (50)

0 .173±o.007 (82) 0 .08±o.OO9 (75)

0 .IOo±o.005 (49) 0 .05±o.OO9 (50)

0 .083±o.005 (40) 0 .04±o.OOI (35)

0.029 0 .016

duc ing the enzyme activity the samples spoiled within four weeks of storage due to microbial spoilage.

Samples treated with HR, sulphites , and BLE did not show much sign of spoilage during the storage. For­mu lation containing BLE was most effective in reduc­ing both the bacterial (Table 4), and yeasts, and mould counts (Table 5). Thi s formul ation was also responsible in maintaining the original texture of samples without much softening.

Of the va rious families of resorc inol derivat ives, HR has proved to perform well in preventing the shri mp melanosis2.1, in enzymatic browning in apples l U I and mushrooms25

. It has also been shown to exhibi t no tox­icity26 . The synergisti c mechani sm by which AA en­hances HR inhibition of PPO and browning is not prop­erly established although AA may act by reduc ing 0 -

quinones produced by phenolases not inactivated by HR or by reducing any oxidi sed HR.

BLE alone could also help in main taining the tex­ture to a larger ex tent. In southern parts of India, Banana leaves have been in use traditi onally as a packagi ng

392 J SCI IND RES VOL 59 MAY 2000

Table 3- Inlluence of different treatments on polyphenol oxidase activity after storage for four weeks at O"C

Treatments

T-I

T-2

T-3

T-4

T-5

T-6

T-7

T-8

T-9

T-IO

CD 5 per cent

Apples

31.40±0.32

20.23±0.84

31.3O±O.55

26.96±0.78

19.40±0. 17

16.46±+0.42

16.90±0.43

24.90±0.76

16.30±0.28

12.30±O.43

2.044

Polyphenol oxidase activity (units g-' Lwt )

Mushrooms Potatoes

93.0o±2.36 7.50±0.29

55.3o±l.47 4.00±0.48

83.6o±2.84 7.20±O.31

74.3o±2.13 6.10±0.34

50.56±3.15 3.80±0.24

42.2o±2.30 3. 50±0.23

49.6o±O.74 3.9o±+O.12

74.8o±2.73 6.10±0.34

48.2o±O.83 3.40±0.15

36.lo±1.80 2.90±OO8

15.974 1.053

Table 4 - Total bacterial counts on apple, mushroom and potato slices after storage for two weeks at O"C

Treatments

T-I

T-2

T-3

T-4

T-5

T-6

T-7

T-8

T-9

T-IO

CD 5 per cent

Apples

28 .o±3.20

9.o±1.04

12.o±l.20

15 .o±1.1O

7 .0±0.64

5.0±0.17

6.o±O.32

10.0±0.42

6.0±0.20

2 .0±I.07 3.469

Counts [c fu / g (x 10") 1

Mushrooms Potatoes

36.5O±O.65 16.0o±O.85

12.75±0.32 5.00±0 .20

18.0o±O.40 8.00±O.32

20.0o±O.20 11 .00±O.42 10_25+0.32 3.75±O.32

7 .00±0.20 3.00±0.20

8.lo±O.43 3.75±0.32

13.75±0.32 5.75±0.32

8.50±0.64 4.00±0.53

4.0o±O.20 I.OO±O.I O 1.163 1.098

Values are means ± SEm

Treatments

T- I

T-2

T-3

T-4

T-5

T-6

T-7

T-8

T-9

T-IO

CD 5 per cent

Values are means ± SEm

Table 5 - Yeast and mould counts after storage for four weeks at O"C 2

Counts( c.LlI /g (x 10 »)

Apples Mushrooms

1.50±0.02 2.00±0.10

0.7I±O.04 0.93±O.O3

0.74±O.O4 1.00±0.08

0.81±O.03 1.18±0.03

0.6I±O.05 O.83±0.O3

O.50±O.04 O.65±0.06

0 .53±O.O4 O.63±O.05

O.59±O.O3 1.00±0.04

0.35±O.O2 0.55±0.02

0 .24±O.O2 0.35±0.O5 0.130 0.336

Potatoes

I.02±O.07

0.47±O.03

0.52±O.03

O.58±O.03

0.4 I ±O.04

O.27±O.05

0.27±O.O4

O.44±O.05

0.25±O02

0.15±O.02 0.149

KAUR & KAPOOR: ENZYMATIC BROWNING 393

5 ~ ~ MUSHROOMS

• • APPLE'S .

E

'" '" p 0 POTA~OES

-:' a: :x: .j

l « ::> 0 Vi UJ c:r: 2

1

' 0 0 4

STORAGE TlME (WEEKS)

Figure I - Residual 4-hexylresorcinol as a function of storage time at O"C

material for keeping the food fresh. Probably there is some natural chemical compound present in its leaves, which accounts for its preserving property. The results of the present study indicate that formulations contain­ing BLE were most effective in restricting browning in apples, mushrooms, and apple slices. Besides the tex­ture, taste freshness, and quality of frozen products were also maintained to a greater extent. The ability of BLE to inhibit enzymatic browning in fruits might have re­sulted from the ability of this extract to form complexes with PPO substrates, thereby preventing their oxidation to quinones and subsequent polymerization to brown pigments. Further studies need to be can'ied out to opti­mize the conditions to achieve best results with BLE and also to find out the factors responsible for its better and added contribution for maintaining the quality of frozen products.

Gradual loss of product firmness on storage was observed in all the treatments. The loss of firmness is a gradual process of catabolism of fruits catalyzed by dif­ferent enzymes and is accelerated by cutting and slicing. Treatments like AA, HR + AA, sulphites, and CaCI , although reduced the loss of firmness to a larger exterYt but did not prevent it completely. However formulations containing BLE ( T-8 to T-I 0) proved to be the best.

More likely the residual content of HR in different treatments and refrigerated samples may exceed one ppm

and this may lead to certain objections. In the present study, residual levels of HR in the treatment (T-I 0) re­duced to almost 0.75, 0.30, and 0.125, respectively

----(Figure I). Thus the results also suggest that it is safe to use HR as an anti-browning agent.

Conclusions

Formulations consisting of HR, ascorbic acid , and BLE proved to be the best in maintaining the quality of apples, potatoes, and mushrooms during storage.

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