STUDY ON WHITE PEPPERCORN PRODUCTION BY VISCOZYME® L AND PECTINEXULTRA ® SP _ L
Le Hong Phu'". Le Thi Hong Tuyef', Le Nguyen Quynh Tram\ Ngo Huynh The Hai' ' International University. Vietnam National University, HCMC
University of Natural Resources and Environment, HCMC
SUMMARY
CeUulase and pectinase are the most common enzymes tt*ich used in husk removal of black peppercorn. They can break down pectin and cellulose in a shorter time and more safety ttian odier mettiods. This is &e best way because ttie effective is so high, saving cost and dme, no polluted as traditional mettiod. This smdy qjplied Viscozyme _ L and Pectinex Ultra SP_L, die commercial enzymes, to produce white peppercorn which have high qualiy, aroma and pungency are improved and safety than traditional methods. While Viscocnzyme_L took 8.5 hours to remove 100% husk in black peppercorn with 2.67% enzyme and dried in 90''C for 35 mm con^iare to 9 hours with 5.33% enzyme and dried in WC fiw 40 min by Pectinex Ultra SP_L. The piperine in treated peppercom was 5.3-5.5 higher than in raw material (3.9). The quantity of piperine was ttie highest when treated by Viscozyme_L.
ViscozymeL and Pectinex Ultra SP_L did not release aflatoxin during treatment White peppercom was produced from them can be used as food for human without toxic All results were testing at Quality Assurance & Testing Center 3 (31 Han Thuyen, Dist 1, Ho Chi Minh city, Vietnani). From ttiis research, it can be concluded that Viscoenzyme_L and Pectinex Ultra SP_L can be spiled weU in white pepper processing industry widi high quality, safe production and shorten processing duration than tradidonal method.
Keywords: black peppercom, cellulase, enzyme, pectinase, treatment, white peppercom, Viscozyme _ L, Pectinex Ultra SP_L INTRODUCTION
Peppercom is a king of spice Uiat improve smelt and taste of meal. Original pepper plant is in the Malabar Coast of India (Kumar N. ,2003), It was popular in tropical countries. Vietnam, India, Brazil, Indonesia and Malaysia are major peppercom producers in tfie world ( JeromI and Ramanatfian, 1993). Vietnam has become ttie largest producer of peppercom in the world since 2003 (Almin and Eriksson, 1967). However ttie price of Viettiam's peppercom is lower than one of ottier counties. Black peppercom has tieen major product in Vietnam, however the price of white peppercom in 1.5 fold than black peppercom (Vanin, 2005). White peppercom can't have high quality production in Vietnam, since it is produced by traditional method. Peppercom is treated by enzyme which Is a new mettiod to produce white peppercom.
The basic of this method is the use of specific enzymes or microorganisms to degrade large molecule to small ones in peppercom. Pectin and cellulose major components in cell wall of peppercom are difficult breakdowi by physical methods. (BeMiller, 1986)
Mil
MATERIALS AND METHODS 111 j Materials
1111 Black peppercom was supported by Son Thanh company, Phu Yen province include 94% ripening peppercom with 1111 black, light brown color, 6% of peppen^om is still young and spoiled size of pepper is 0.2 cm for each. All peppercom I I I I were kept In the dried place at room temperature (preventing direct light and risks of contamination) for usage of whole
experiment.
Commercial enzyme - Viscozyme® L and Pectinex ® Ultra SP_L were provide by Nam Giang company, 133/11 Ho Van Hue, 9 ward, Phu Nhuan Dist, HCMC.
Methods
Determination of the cellulose content by using strong acid: 1 g of b\adk. pepper powder after being dried at 100°C till n i l reaching a constant weight was put into 500 ml flask. Adding 16.5 ml of add from a mixture containing 15 ml concentrate
nitric acid and 15 ml concentrated acetic add into the flask, trailed for 30 minutes. After that, the solution was cooled down and diluted with dHzO. (Mui, 2001). Next, the solution was filtered by using a filter paper witti known weight, precipitated cellulose was first wished with 15 ml of dHzO in several times and then 96% ethanol in ttiree times. The filter paper containing predpltate is dried at 100°C until reaching the constant weight, the quantitative cellulose (C) is 1111 calculated by
Where C: the quantitative cellulose. A: weight of precipitated cellulose (g). S; weight of sample (g)
Oetemninatlon of the pectin content by using calcium pectate method: 0.15 g of black pepper powder is mixed witti 100 ml NaOH 0 1N, the mixture is kept for 7 hrs or ovemight for converting pectin into pectin acid. Adding 50 ml of CH3COOH 0.1N to the mixture, after 5 minutes continued to add 50 ml of CaClz 2N (Mui, 2001). The solution remamed in an hour, then boiled more 5 minutes and finally filtered by using filter papper. Predpltate on the paper was washed several times with hot water; using AgN03 1% to know the presence of chloride ion in waste water, when those ions had been removed, the filter paper which was containing precipitated calcium pectate was dried in the oven at lOS'C until its Weight was constant The quantitative pectin (Pt) was calculated as
Where S: weight of sample (g). B: weight of precipitated caldum pectate (g). 0.92: coefRdent of pectin contained in i ( of precipitatton
Checking pectinase activity by using Spechophotography mettiod: 1 g of product from Viscoenzyme _ L and PecSu Ultra SP_L was grinded witti 50 ml of HzO wthin 5 minutes. The suspension is filtered by cloth and cotton to elimiriBie solkJs. The crude enzyme solution is ttien diluted into 1000 times in oreler to test pectinase activity (Phu, 2009) 5,„
CTude enzyme solution after dilution of 1000 times transfered into a new test tijbe containmg 10 ml pectin solution }\
shook ttie hJbes well and then incubated at 30° C in 60 minutes. After ttiat the solution was added with 2 ml of ZnSa 15%, shook and filtered ttiem. They filtered solution was 5 times diluted. Next, 2.5 ml of diluted solution was transfetrai to a new hjbe, added 5 ml of anttirone, shook well; ttien incubated ttie solution at 70" C in 12 minutes, finally coctel down at RT to measure OD witti ttie wavelength of 584 nm (by using spectrophotometer). The pectinase actiwty (P) ^ calculated by using the folbwirig equation:
0.34 X &0D - 0.0104 p = ^ n
Where A OD. optical density of sample after reaction writti enthrone, V: amount of enzyme (g or ml), D: a dilution factor Checking cellulase activity by using CMCase: 1 g of product from Viscozyme® L and Pectinex ® Ulti-a SP_L was mixj witti 50 ml HzO wittiin 5 minutes. The suspension was filtered by cloth and cotton. Then, 0.5 ml enzyme was diluledij dtrate bufter, after ttiat put it into a test tube At least two dilutions must be made of each enzyme sample investigaled]
One dilution should release slighUy more and on slightly less than 0.5 mg (absolute amount) of glucose (= reduonj sugar as glucose) in the reaction conditions (Mui Nguyen Van, 2001, Phu Le Hong , 2007). TTie tubes were added 0.5 iif subsfa'ate sdution, mixed well and incubated them at 50°C for 30 minutes. Added 3.0 ml DNS into solution, mixed m All samples, enzyme blanks, glucose standards were boiled in water baUi for 5 minutes. After boiling, put all sample iii cold water sink, added 20 ml dHzO, mixed solutions by completely inverting the tube several times. And finally, i samples were measured OD at S40nm (by spectrophotometer). The cellulose acti'vtty determined by CMCase i calculated by equation
Enzymeeoncentratlontorelease D.Smggluc ' ( unit ml" )
Testing piperine and aflatoxin of peppercom Testing piperine and aflatoxin of products: black pepper as control sampi and white pepper after incutiaton were tested aflatoxin and quantified piperine at Quality Assurance & Testing Center;
(49 Pasteur, district 1, Ho Chi Minh City, Vietnam) with AOAC 2007 (991.31) and AOAC 2007 (987.07) methnS respectively
Treahnent: 100 grams of black peppercom was soaked in water at a flxed moisture content (60%) after ttiat removmi excess water. Viscozyme _ L and Pectinex Ultra SP_ L were added at the ratio (%) of 0.33, 0.67,1.33, 2.67, 5.33,10.61 21.33. Mixed the enzyme and peppercom was well. Example of Viscozyme _ L was kept in 2 4 6 8 10 (hours) al4[l- 45 C. Example of Pectinex ® Ultra SP_ L kept at 20 - 25°C. The factors of research were soaked time, moisture conleit
ratio of biopnaducts and the ti-eatment time shown in result section. I RESULTS AND DISCUSSION
Optimum the moisture content for incubation
30 40 45 50 55 50 65 70
Moi stiFecortertol peppercorn overtime (%) Figure 1 Percentage of skin removal to moisture content
Me ™o™, nmH T ^ ' T " " " ' " ' ^°''' "' ' " " ' ' • " ' ^ ' = ° * ' " "hite Pectinex UIM SP^
iiie Deooar nmni irtinn Tha mni^h.... . •_._ .. 1/1,5 I sang ..™ ™™.,™ . . , „ . „ , . aarne mnnmnn. I, J J V , ^^sy 10 peel the pepper husk by the resolutiotl i
; e e l ^ ™ r s 9 : 4 % o 7 i ™ p e " p e X ^ ^ ^ ^ ^ ^ I " ™ ? " ^ " '•' ^ '"•^^ "== '^ ""^
samples f o l l o i . i i p a t 8 1 % o r S ™ p 9 o ™ , n m l S T h ^ r ^ ^ ^ ^ - 7 1 p e p p e ™ m , ^ s r e m o v ^ v e ^ d S t r n S o u n l n f i ^ ^ ^ ^ " ^ ™ ' ' ^ " " ' " ' " ^ " " ^ "=='"'9' "^ " " ^ i
»uld be amoved tier, p e p ^ ™ m Sy w ^ r a b s l t on n r n ' " ' ' ! ? ' ^ "^ "Sf""^" ™'>' ' " = • '"'"^ 1 « - ™'J3 rorce. When hun„dHy »as 7 ^ , Ihe p ' J ^ ^ e p ffL'^o^a 3 i St^^^^ ^ ' ' "^ " ^ >^ '"^«' "' " ^
.iC CONG NGH^ SINH H y U I UAN OUOC 2013
Optimum ratio of enzyme for the treatment
Two commercial enzymes were used, Viscozyme_ L and Pectinex ® Ulb^ SP_L. The husk of peppercom was removed increasingly with a rising volume of ^izyme. The optimal moisture content of peppercom and duration for termentetion were kept constenL The ratio of enzymes (%) were calculated by below equation:
weight of enzyme ( j ) 100 g black peppercom for treatment
^
0.33 0.67 133 2.67 5.33 10.672133 R<tkiofenzyine(%)
Figure 2: The removing totally husk from peppercom
According the graph, black peppercom was treated within 8.5 - 9 hours and double addition enzymes every time of treaUiienL From 0.33% of enzymes, the percent of husk removal was very low, just 19.3% of white peppercom was produced. Pectinase and cellulase still produced but not enough to remove the pepper skin. White peppercom was produced 94.6% with only 2.67% of Viscozyme _ L. In ttie ottier hand, 5.33% of Pectinex Ultra SP_L produced 93.8%
white peppercom.
150
50
0 -
f
120
0 ^
240
^
360 5 5 * -
4S0 6D0 720
' • ^ V Sen zyme_L
• " Pectinex
* Control
Duration lor treatment with eniyme (mins) Figure 3: The removing totally husk from peppercorn
The duration affected clearly to the b^atment quality. The pectinase and cellulase catalyze pectin and cellulose, two substrates in peper skin and the treatment may not bring good economics aspect wth long duration and if the duration is too short, the white produced pepper does not bring good result because of no all skin removal. The white peppercom was produced more Uian 95.6% by Viscozyme_L. The remaining 4.4% of peppercom was young, flat and bad which cannot use in meal. Pectinex Ultra SP_L had the weaker eftect to remove the husk from peppercom, only 91.3%. In a short duration of treatment (< 8 hours), enzyme did not have enough time to degrade all pectin and cellulose in peppercom. Theretore, husk of peppercom remained <vi it and soluble solids could not be extracted from cell of peppercom. On the other hand, it could release toxic such as Aflatoxin which causes hepatic carcinoma in human if Ihe duration of tiBatment was so long (> 10 hours). The optimal duration of hBatinent vtras detemiined vAxen totally husk was removed from peppercorn, high soluble solids and do not have toxic.
The effect of temperature on dry white peppercom was very important. If the temperature was so high (> 100°C), it can bum tiie peppercorn easily. The peppercorn can't be dried and content high humility. In tiiis research, the temperatijre was used to dry white peppercom around 90°C in for 35 min. The duration of dry and the temperature must be suitable.
The color, soluble solid and preservation of white peppercom can be effected by temperature. If ttie duration was so long, the peppercom can be burned and the color was so dark. That was not good for sensory.
Table 1, Summary of treatment by using enzymes
Products Moisture content (%) Ratio (%) Treatment duration (hours) Percentage of husk removal [%) Viscozyme 60 2.67 8.5 100%
Pectinex Ultra SP_L 60 5.33 9 100%
Piperine (C17H19NO3) Is an alkaloid which is responsible for pungency of black peppercorn. Piperine has useful effects on human health including enhance ttie bioavailability of various supplemental nutiients. It is known as a cenb^l nervous system depressant and has good anticonvulsant, antimicrobial and insecticidai properties( BanoG, AmIaV, RaniaRK, Zutshiu and Chopra CL., 1987) .Raw material (black peppercom) and treated peppercom were diecked quantitative piperine by QUALITY ASSURANCE & TESTING CENTER 3 ( 31 Han Thuyen, Disl 1, Ho Chi Minh city, Vietnam). Result of piperine shown on the table 1.2
Table 2: The quantitaUve piperine i n peppercom
Raw matera (tilack peppercom) Peppercom bested by Viscozyme L Peppercom treated tif Pectinex Uttra S P _ L
Quantitative pipenne Mettiod I
; AOAC (987.07)
Aflatoxm is natural mycotoxins ttiat are produced by many species of fungus, m o s t n o t a b l y A. flavus a n d A. parasiticus It is high mutagenk; and towc to human such as abdominal pain, pulmonary e d e m a , convulsions, c o m a ( A d a m . M R. and Nout .M.J.R. 2001) Raw material ( black p e p p e r ) and treated p e p p e r c o m witti V i s c o z y m e _ L a n d Pectinex UHra SP L was tested Aflatoxin QUALITY A S S U R A N C E & T E S T I N G C E N T E R 3 (31 Han T h u y e n , Dist 1 , Ho Chi Minh ci^, Vietnam).
Table 3: The result of Aflatoxin testing in peppercom
Raw malenal (black peppercom) Not detected Peppercom treated by Viscozyme L Not delected Peppercom treated by Pectinex Ultra SP _L Not detected C O N C L U S I O N S
This stody w a s earned out to find the best conditions for Viscozyme _ L and Pectinex Ultra SP_L activities. T h e resulb were resent as ttie highest activity of commercial enzymes, after 8.5 - 9 hours, w h i t e p e p p e r c o m w a s produced by using Viscozyme _ L at 40''C, pH 4.8 and Pectinex Ultra SP_ L at 25°C, pH 5.0 and kept optimal moisture at 7 0 % . Finally, the tiBatment of Viscozyme_L has a better production if comparing the economics s i n c e tiie Pectinex Ultra S P _ L needs with percentage of peel at 100% .Vlsoozyme_ L can b e used to produce white p e p p e r c o m with a safety, high quality and good sense.
REFERENCES
Hunt AC (2004). Pepper From Viet Nam' Quality Makes A Difference. Inlemational Trade Center, Switzerland
Almin KE, Erfltsson KE (1987).. Enzymatic I. Viscometrlc mettiod degradation of polymers. For the deterniination of enzvmatic activity
p.238- 247 "
Adams MR, Nout MJR (2001). Fennentation and food safety. An Aspen publication, Maryland
Bano G, AmIaV, RainaRK. Zutshlu. Chopra CL(1987). The effect of piperine on phamiacokinetics of phenytoin activity. 238-274 BeMiller J (1986). An introductfon to pectin and cellulose: stnjcture and properties. 3-11
Naveen K (2003) A Report of Black Pepper (Piper Nigmm L) Scenario .S,L: University of Agncuture Sciences, 83: 3.
S ? I S i r « S v S s T ? " * * ^ ^ ' ' " " " ^^^' ' " ^ * * " ^""^ ' " ' " ^ ' '^ ^ " ^ ' ^ ' ^ ° ' 3 " ^ ^ " ^ '"stabilily,lndian Joumal of agrtcultura
^•2^\uiS^^^' ^^^^ "^ ^ ' ^ ' ^ ^ " ^ ^"^""^ "^ '"^*"* ^^^^^' ^''^^"' ^ ^ P " ^ ' Assiociation. 2010. Report on executive board meetmi
NGHIDN Cl>U S A N X U A T T I ^ U SO B A N G VISCOZYME L AND PECTINEX ULTRA SP _ L
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