heat stability of lipoxygenase and peroxidase during...
TRANSCRIPT
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
..
KAUR et al. : BLEACHING OF VEGETABLES 1007
w
'" " % W .., ,. )( o Q.
~
oL-____ .-______ .-____ -i _____ -~-~-x~-~-~-~--=-=-~ a
M'INUTES
250
200
ISO
lOa
50
~
~
'", 'c " w V1
" 0 ;;: 0 cr w ..
Figure I-Comparison of enzyme inacti vation models with peroxidase(---) and lipooxygenase( --) during conventional water bl anching(BW) at IOOIlC(e) and microwave( MW }
blanching(x) in biller-gourd. Vertical bars represent average standard deviations ( N= 3 )
-"' V> <f Z
·W
'" ;; 2 o .. ::;
' J
""IN UTES
50
200
., ,~
150 '. " w V> <t e
100 " 0 0: W Q
50
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.
~
~
c " w
'" " z w
" ,. x 0 "-:::;
MINUTES
00
c
" w
'" 40 ~
10
X o c: W Q.
Figure 3-Compari son of enzyme inacti vation model s with peroxidase(---) and lipooxygenase( --) during conventional water blanching at I OODC( eland microwave blanching(x) in peas. Vertical bars represent average standard deviat ions (N=3 )
, I
\ 10
-'i " I'
'D 8 ,I
" J!! " i:
" -"' 6 VI ..: %
' W <!> ,.
4 )(
0 .. :;
o
" " ,I ,I, " " ",
... ---
MINU1ES
12
10
s 'i
-'" V1
'" o 4 -x
o cr !lI
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.
References Floyes S, Perera C. Chevis P & Holden J. Some Pro perlH.:s of Lipoxygen<1 se in M atu re(cdibl e) Corn (Z <'fI .'lIm ·s) and It'. IlIacti vat ion by Microwa ve. Pmc A gHili So(' o(N Z. 22 (I t)0::: ) 0- 15.
2 Boyes S, Chcris P, Holden J & Perera C. Microwave alld Water Blanching otT om Kernels: Cont rol of Uni lorJ nity 01 Ilcat -
1
KAUR e l al. : BLEACHING OF VEGETABLES 100Y
ing During Microwave Blanchin g, J Food Process Preserl'. 21
( 1997) 46 1-484. 3 Willi am D, Lim M, Chen A, Pangborn R & Whitaker J. Blanch
ing of Vegetables for Freez ing- Whi ch Indicator En zy me to Choose, Food Technol. 40 ( 1988 ) 130 .
4 Barrett D & Theerakulkait C. Quality Ind icators in Blanched Frozen Stored Vegetables, Pood Technol, 49 ( 1995) 62-65.
5 Gavanth orn C, Nagel C & Power J, Thermal In acti vation of Asparagus Lipoxygenase and Peroxidase. J Food Sci, 56 (1991)
47 - 50. 6 Thomas R L. Jen J J & MOlT C V. Changes in Soluble and Bound
Peroxidase, IAA ox idase During Tomato Fruit Development, J Food Sci, 47 ( 198 1) 158- 16 1.
7 Chen A D & Whitaker J R, Purification and Characterization of Lipoxygenase from Im mature Engl ish Peas, Food Res, 20 (1986) 480-482.
8 Pelletier 0 , Vitam in C, L-ascorbic Acid and Dehydro-L-Ascorbic Acid. in Melhods ofVilalllin Assay , edited by J Augustin , B P Kl ein and D Becker (John Wiley and Sons. New York ) 1985.
9 Carotene Panel of Subcommillee on Vi tamin Estimation .:Rap id and Accurate Method for Estimation of B-Carotene, In Melhods of Plan I Analysis . ed it ed by Peach K and Tracey M (Springer- Verlag, Berl in ) 3 (1955) 308 .
10 Baard Seth P . Enzymatically Induced Quality Changes in fresh Frozen Carrot. Ac{({ Horl. 93( 1979) 67-74.
II McDani el M, Mon tgo mery M. Latham D & Lundahl D. Eva luation of Hi gh Temperature -Shon Time (HT ST) Blanched Frozen Sweet Corn , J Food Sci, 53 ( 1998) 760-764,853.
12 Pi zzaro F, Aggujaro R & Bertolo G, Cineti che d' lnalli vzione Termica DegJi Enzimi Ossidas ici Dell a Carata , Rev Sci A lIill/ ellf Anno, 22 ( 1993) 279-285 .
13 Klein B P, Fruits and Vegetables, in Food Theory and Applica lions, edited by J B0wers (Macmillon Publi shing Co. ew York) 1992.
14 Guines B & Bayindrh A, Peroxidase and Li poxygenase In act ivation During Blanching of Green Beans. Green Peas and Carro ts, Food Technol, 26 ( 1993) 406-410.
15 Chen 13 H & Han L M, Effec ts of Different Cooling Methods on the Yie ld of Ca rotenoids in Water Convo lvulu s (//J () lIwea
aqualica), J Food Pml, 53 ( 1990) 1076- 1078. 16 Chen B H, Studi es on the Stab ilit y ofCarotenoids in Garland
Chrysanthenum (lpollloea SIJP. ) As Affec ted by Microwave and Conventional Heating. J Food Pml, 55 ( 1992) 296-300.
17 Halpi n BE & Lee CY, Effects of Blanching on Enzyme Activi ty and Quality Changes in Green Peas. Food Sci. 52 (1987) 1002-
1005.