Journal or Scient ific & Indus tri al Research Vo1.5 8, December 1999, pp 1005- 1009

Heat Stability of Lipoxygenase and Peroxidase During Blanching of Vegetables

Charanji t Kaur*, Sushi I Kumar and Hari sh C Kapoor**

Division of Biochemistry, Indian Agricultural Research Institute, New Delhi 11 0012, India

Received: 2 1 Jul y 1999; accepted: 02 September 1999

French beans, carrots , peas, and bitter-gourd were blanc hed conventi onall y and in mi crowave ror different dura tions. Lipoxygenase and peroxidase were taken as indicator enzymes to test the adequacy orblanching. Lipoxygenase was found to be more heat stable than perox idase. Quality retention of processed vegetables was round to be hi ghest in microwave blanched products. Results suggest th at the use of peroxidase is to be continued as indicator enzy me, instead or li poxygenase, as suggested by some recent research.

Introduction Usually the raw vegetables cannot be stored for

longer periods, even at -200 e , because of certain

quality changes catalyzed by some enzy mes. These enzy mes primarily responsible for quality deterio­ration of raw un blan c hed vegeta bl es are: lipoxygenase (LOX), Peroxidase (POD), lipase, catalase, and proteases, e tc. LOX can cause off­flavour development and cata lyzes the fo rm ati on of fatty acid hydroperoxides from polyunsaturated fatty acids . The breakdown products from these reacti ons have been implicated in aroma and taste changes in many processed foods 1.2 • POD, to a lesser extent ,

can cause colour changes while ascorbic acid oxi­dase and transaminase may resu lt in nutriti ona l changes' . Blanching is the process associated with inactivati on of these enzymes and is used primaril y to maintain sensory and nutritional characteristics of frozen products. Most of the studies done so far have shown that POD is most heat stable enzyme and therefore bl anching done to the extent th at this enzy me is lost leads to inac ti vation of several other enzymes which are otherwi se responsibl e fo r main­taining the quality of blanched products ' . Barrett and Theerakulkait~ , on the other hand , have recom-

* Division of Fruits and Horticultural Technology

**Correspondi ng author

mended the use of LOX as an indicator enzyme be­cause acco rding to them its des tructi on durin g

blanching may not affect the ac tivity of other en­zy mes. They report that POD is more heal stabl e than LOX, However, they have also indicated the limitations in its use because of non-avail ab ility of any simple methods for its assay. Recentl y, it has been shown in Asparagus tips that LOX is more heat stable than POD under blanching condition s, for some selected vegetables5

. These authors have ad­vised independent evaluation of different foods fo r their LOX and POD ac ti viti es . In the present study, an attempt has been made to reevalute the adeq uacy of using LOX and POD as indicator enzymes for optimizing blanching conditions for some se lected vegetables .

Materials and Methods Sample Collection-Bitter-gourd (Momo rdica

chara~fia), French beans (Phaseolus vulgaris) , Peas (Pisum ,iativltll1 ), and carrots (Daunts carota) were purchased from the local market, washed thoroughl y with tap water, rinsed in distilled water and then cut into 2-3 cm pieces in a French cut sty le . An ordi ­nary water bath mainta ined at loooe was llsed as water bl anching equipment (BW). For microwave (MW) blanching, vegetables were taken in a corn­ing bowl and pl aced in a domestic Microwave oven

]006 J SCI IND RES VOL.58 DECEMBER 1999

(BPL Model BMO-700T, 650W) at high power level (2450 MHz) and blanched for different durations. After blanching, the samples were immediately cooled in ice water, drained well and packed in polythene bags which were sealed after pressing out the air and stored at - 20°C

En zyme Extraction

Vegetables (lOg) were homogenized in cold 0.05 M phosphate buffer (pH 6.5) containing 0.5g poly­vinyl polypyrrolidone and acid washed quartz sand. The homogenate was centri fuged at 10,000xg for 20 min . The supernatant (Crude extract) was col­lected and used for measuring enzy me activity.

Peroxidase Assay POD (ECI , 11 .1.7) act ivity was measured as the

change in absorbance at 470nm using guaicol and H,O, as substrates6

. The substrate sol ution of 0.5per ce-nt -(v/v) guaicol in 0. 1 M phosphate buffer (pH 6.0) was stirred fo r 30 min. , 0.008per cent (v/v) 30per cent H,O, was added immediately before use. Crude extracts (50~ J) were added to 2.5-ml substrate in a cuvette and absorbance was recorded . One uni t of POD activity was defined as change in absorbance of O.l/min .

Lipoxygenase Assay Method of Chen and Whitaker7 was fo llowed for

assay ing the LOP (E.CI .13 .11.12) activity. A 0.01-M stock solution of linoleic ac id in I.ON NaOH and Tween-20 in distilled deionised water was prepared afresh . Before assay the substrate stock ."ol ution was diluted 5-fold with 0.2M phosph ate buffer pH 7.0. The substrate -buffer solu ti on was fl ushed wi th air for 10 min and allowed to equili­hrate at 25°C for 10 mi n before use. For the assay I OOul of crude extracts was added to a 3.0mlli noleic ac·id substrate in a cuvette, mixed and absorbance recorded at 234nm over time. One unit of enzyme activi ty is defined as that amount of enz me, which produces a change of absorbance of I.O/min .

Ascorbic Acid Analysis. Ascorbic ac id was determined by usi ng the assay

as desc ribed by Pelletier~ . Samples ( lOg) were ex-

tracted with 6 per cent metaphosphoric ac id and titrated against 2,6, dichlorophenolindophenol dye.

f3- Ca rotene It was estimated by using the method described

by Carotene Panel of Subcommittee on Vitam in Es­timation9

.

Results and Discussion Many of the quality changes, that frozen veg­

etables fall victim to, are catalyzed by enzymes and so it is logical to choose an enzyme as an indi cator of adequacy of blanching process . In the present study, a re-eva luar ion of the use of POD or LOX as indicator enzyme for optimizing the blanchi ng con­ditions has been attempted by taki ng di fferent veg­etables like French beans, carrots, pea. , and bitter­gourd . Results show qu ite high activ ities of these enzymes in raw unblallched vegetables. Conven­tional blanching in water (1 00°C) (BW) shows grad ual declines in POD, as well as LOX act ivities with time. A 3min treatment resul ts in around 75per cent loss in bitter gourd (Figure I). 70per cent in french beans (Figure 2). and in peas (Figure.]) and 80 per cent in carrots (Figure 4). POD activities . For LOX the losses were around 45 per cent in bitter-gourd (Figure J), 55 per cent ill french bean (Figure 2), 60 per cen t in peas (Figure 3) and 42 pe r cent in ca rrots (Figure 4). A blanchi ng ti me of 5 min resulted in substantia l losses in the POD or LOX activities in all the vegetab les . bu t hearing for longer periods may result in more se vere deteriorat ion in nutritional quality of the products. Data wit h the MW blanch ing on POD and LOX reveals that in MW blanchi ng the time required to reduce equi valent or even more amount of POD and LOX acri viti es is less as compared to BW blanching. A bl anching time of 3 min is sufficient to induce around 80-90 per cent loss in POD and 60-70 per cent loss in LOX activiti es (Figures 1-4).

Conventionally the vegetables arc blanched to the poi nt of dest ruction of POD acri vit y and it is genei"­ally accepted lhat if POD is destroyed then it IS quite likely that other enzymes will not surviv '. The use of LOX is, therefore, reco mmended by barett amI

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blanching(x) in biller-gourd. Vertical bars represent aver­age standard deviations ( N= 3 )

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Figure 2-Compari son of enzyme inactivation modcls with pcrox i­dase(---) andlipooxygenase( --) dUling conventional wa­ter blanching(BW) at I OOOC( e) and microwave blanching (MW) (x) in french beans. Vertical bars represent average

standard deviations ( N=3 )

Theerakalkait4 as an alternative to POD. They have suggested that rather than designing a process that inactivates all the enzymes or the most heat res is­tant ones, it is better to select an enzyme that is di­rectly involved in major deteriorating changes dur­ing frozen storage. But it has generally been ob­served that there is no single enzyme that is respon­sible for all quality changes during storage.

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Figure 4--Comparison of enzyme inactivation models with perox i­dase(---) and lipooxygenase( --) during conventiona l water blanching at I OO'C( e)and microwave blanching(x) in carrot s. Vertical bars represent average standard devia­tions ( N=3 )

The overall data generated from the present study, on the contrary, suggests that LOX is more heat stab le than POD. Similar results have earlier been reported by Gavanthorn et al.5 in Asparagus. There­fore the blanching done to the point of destruct ion of LOX may result in overall deterioration in the quality of the products. So it is better' to use POD as an indicator enzyme for blanching . Till today the

IOOS J SCI IND RES VOL.58 DECEMB ER 1999

Tah le I- Effect of blanching methods on the ascorbi c acid and P- carotene profile or vegetables

Vegetabl e Ascorbic acid mg/ I OOg Carotene ~g/ I OOg

Control BW MW Control BW MW

French bean 25.2±0.25 15.4±0.13 2 1. 5±O.18 I 22±92 62±5 101 ±8

Bitter gourd

Carrots

70.3±0.48 35.2±0.23 56.7±O.32 120± 10 64±5 95±6

10.3±0. 14 4.3±0.08 8.3±O. 11 8890±85 4308±4 1 7 11 5±68

Peas I 5.4±0. 1 5 5.7±0. 11 13.2±0.12 79±7 35±3 62±6

Val ues are mea n ± SEm

assay of POD is more pragmatic and practical for freezing industry in comparison to LOX assay. In­terferences in LOX assay make its measurement all the more difficult 1o. 12 . Till an easy and rap id method fo r LOX assay is developed it is not poss ible to use it as an indicator enzyme for blanching purposes in frozen storage industry.

The results further sugges t that a blanching time of 3 min in MW at 2450 MHz is adequate to achieve optimum blanching conditions without much loss in qual ity of processed froze n products. This is be­cause MW blanching is more efficient in inactivat­ing enzymes at a temperature not enough to affec t the ,-j ;' ali ty of the products to a greater extent. Ascor­bic ac id , p-carotene, colou r, texture, and taste were takcn as parameters for quality determination of bl anched roducts.

In vegetables, ascorbic acid is one of the most lab ile nutrients because it is readi ly oxidized, water sol uble, pH-, !ight-, and heat sensitive and affected by naturally occurring enzyme system ascorbic acid oxid ase l1 . Average ascorbic acid content of unblanched raw French beans, bitter-gourd , carrots and peas was found to be 25 , 70, 10, and 15 mg/ 100g fresh wt of samples, respectively. Conventional heating at 1000 C for 3 min resulted in 50 to 60 per cent losses (Table I ). MW blanching (3 min), on the other hand , helped in retaining about 80-90 per cent of the ascorbic acid content. A hi gher retention of ascorbic acid has earli er bee n reported in MW blanched green beans and carrotsl ~ . Most like ly, rcduccd bl anching time, less heat, and less water

required for MW blanching produces far less dete­riorating effects.

Simi larly, lower retention of p-carotene in con­ventionally blanched samples, in compari son to MW blanched ones, was also observed (Table I ). Chen and Han l) and Chen 16 have earlier reported similar results. Sensory characteristics such as colour, tex­ture , and taste are affected by blanching conditi ons. During storage at -200 C for th ree months no appre­ciable change in colour, texture, and tas te of frozen MW blanched vegetables was observed . The fro­zen products (MW) were better than the frozen (BW) blanched product. Halpin and Lee l7 , Guines and Bayindrhl4 and Chen 16 have earl ier reported si mi lar results.

Conclusion Thus the above studies suggest that POD shoul d

be continued as an indi cator enzyme, since it is easy to assay and more heat sensiti ve than LOX. However, independent analys is of the spe­cific vegetables is required to ascertain its adequacy. The studies furth er suggest that MW blanching could be considered as more affe ~ ti ve in inactivat ing cer­tain enzymes responsible for quality deterioration during frozen storage.

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