HOI NGHI KHOA HOC CONG NGHE SINH HOC TOAhi 1
LACTIC ACID FERMENTATION OF PURPLE SWEET POTATO (IPOMOEA BATATAS L ) AND BLACK GLUTINOUS RICE (ORYZA SATIVA L) BY LACTOBACILLUS ACIDOPHILUS AND ITS EFFECT ON ANTIOXIDANT CAPACITY OF THE FERMENTED SOLUTION
Dang Quoc Tuan'. Ly Hong Van Nhi
School of aoleohnotogy, intematonal Universily - ViBlnam Nalinnal University, HCMC SUIMMARY
. .. ,.• „ •i..-,;^, „f fi.r™*,.no mimle sweet potato ilpomoea batatas L.) wA blaclc glutinous nee (OJJKO
• ^ ' ' • ^ ^ ' - ' ' ^ ' ^ ^ ' ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ Z e ^ o f ' S n e n a n o n ptocess on ant.o^d... eapae,,, aad
™,,v. L.) wtlh ^ » " ' ? " J ° i ' l ' ^ " J ^ ^ ^ S "potT.^ (PSP) and blaek glutinous tiee (BGR) were saccharified b , t l .
„,hoca»m emtont of the ' " " ^ » ' f » ^ _ _ ^ ™ ' ' g icoantylase. Saeeltanfied PSP «,d BGR, having r=due«g » | „ ' ^ r ' ' r , ° / 8 ' ? ? ' ° ^ l ? X ^ d . b l r S S w e i sub^eted to'laefe acid fementanon using 1% starter euln,n= .f
^ S ™ e ™ t 3 6 M X 10- crU/mL and 1.12 x lo' CFU/mL viable cell oiunl, tespecovel,. Tie andioeyanm eonlent m the SPS
^ u h o n w r S ^ ° ; ^ S » femtentano^ from 164.69 to 102.07 (mg CydJ-glu-E /lOOg d.b.). Tie s ^ t ^ d ™ solution was siSJ'tic™"? ' " ^ Cvd-3-elu-E /lOOg d.b.). Antioxidant capacity of die solutions before and after ' ^ : : 1 1 Z ^ ^ ^ : ^ ^ U - ^ ^ ^ ^ ^ ^ ^ ^ ^ ' ' ^ ^ ^ " ^ — ^ 8 effct Tl-erefore, i. suggested tb.t PSP S T M R f ™ . e d by LacZp^l^ L i d be used to develop healthy food and beverages with the supplement of viable cells and antioxidant activiliei
Keywords: anttiocyanms, antioxidant acdvity, black glutinous rice, Lactobacillus acidophilus, purple sweet potato.
INTRODUCTION
Plant and root beverages are healthy due to their high nutritional value and presence of bioactive compounds derived from ttie substrates used and during the femienteation process (Duangjitchaioen ef al., 2008) In re«rrt years, ttie interest in anttiocyanins pigments in consumer market has increased due to ttieir possible healtti beneffls ^ dietary antioxidants (Brigers et at., 2010) and their deep purple-red color as an alternative to synttietic food dyes in natural food colorants (Wegener et al., 2009).
Purple sweet potato (Ipomoea batatas L) is considered as a healttiy food additive and potential source of natural food colorants due to high level of anttiocyanins (Kano ef ai. 2005). Black glutinous rice (Oryza sativa L). also possess color substances, anttiocyanins, that belong to ttie flavonoids and were reported to possess a free radical scavenging activity (Okiefar.,2002).
Being enriched wrth probtotic bacteria, femiented products have evolved into one of ttie most successful class of functional foods. Uctic ackl fermentation of sweet potato had been studied wrth a mixed cultures of Streptococcus thermophilus and L^ctobacWus bulgaricus (Wongkhalaung, 1995} and Lactobacillus plantanim (Panda, Ray, 2007).
Latobacillus acidophilus have been applied extensively in food fennentation and processing (Lee ef a/., 2011). Tliis sbaifi has been wdely utilized as a dairy starter culture for their ttierapeutic activrties associated witti an intestinal microbial balance. However, infomiation on Lacidophilus fermentation of substrates rich in anttiocyanins such as purple sweet potato (PSP) and black glutinous rice (BGR) has been limited. There has been littte discussion about ttie effect of fermentation on functional ctiaracteristics of Bis femiented substrates. Therefore, this study is aimed at finding the possibility of lacbc fennentation on antiiocyanin-nch substrates, purple sweet potato and black glutinous rice, to aciiieve highest viable cell count. Also, the effect of fermentation on the antioxidant activrty and anthocyanin content of the fermentation broth is evaluated
MATERIALS AND METHODS Materials
PSP and BGR were purctiased from tocal markets in HCM city and An Giang province, Vietnam, They were stored af 4''C until used. L acidophilus was obtained from the Genus collection of Ho Chi Minh City University of Technology. This strain was pnspagated in MRS brotti for 24 h at 3fC and finally stored at -20°C in MRS broth containing 20% glycerol, before being subjected to fermentation. The a-amylase used was Tenmamyl 120L from Bacillus lichenifonrll (Novozymes, density 1.26 g/mL). The glucoamylase used was Amyloglucosidase EC 3.2,1.3 from Aspergillus mger (Sigma, USA. density 1.2 g/mL).
Saccharification of PSP and BGR
PSP were washed, peeled, slk*d and steamed for 15 min. Steamed PSP were mashed while hot and mixed with disUled water. BGR were ground and mixed with distilled water to get a solution of 10% dry matter. Then the mixture was cooked for 25-30 min. Sacchanfication was carried out at 60°C in an incubator for maximum 90 min using 0.15% of glucoamylase and 0.1% (d.b.) of a-amylase.
Lactic acid fermentation of PSP and BGR
Sacchanfied PSP and BGR were heated to 95''C and held for 5 min to inactivate the enzymes. The solutions obtained from the process were centiifuged to get the dear supematant and sterilized at 121°C for 15 min. Lactic acW
: CONG NGH^ SINH HQC TOAN QUOC 2013
fennentabon wrth L. acidophilus was perfomied at 37°C in facuttative anaerobic condition for 24 h. using 1% starter curtures.
Analytical methods
Chemical composition of raw materials (fresh PSP and BGR) were determined for moisture (AOAC 972.20), protein (AOAC 992.23); arude fat (AOAC 2003.05); cmde fiber (AOAC 962.09); total ash (AACC 08-01).
Reduang sugar was determined by using DNS mettiod. pH and titratable addicity of the samples were measured at room temperature (Lee ef al., 2011). Viatile cell count of lactic ackj tiaderia (CFU/mL) was enumerated by plating mettKXl on MRS medium. Total monomeric anthocyanm content of PSP and BGR were detennined by the specdrophotometric pH differential mettiod (Lee et al., 2005). To measure antioxidant activrty, ttie DPPH radk:al scavenging assay was carried out as descrit}ed elsewhere (Sasaki, Ohba, 2004) with a slight modification. The sample solutions were c^ibiliiged at 1300 x g for 10 min. After that the sample solutions were diluted in ethanol at different ratkis (5%-40%v/v) arxi 2 mL ttie diluted supematant sample was mbced wrth 2 mL of 100 pM DPPH in ettianol. Ettianol (2 mL) wrth DPPH solution was used as blank. These solutions were kept in dark for 30 min at room temperature. The at>soibance of ttie mixture was detennined at 517 nm in triplicate.
Statistical arialysis
The analysis of variance (ANOVA) was conducted using SPSS version 16.0 to test the significant difference between groups ( p < 0.05).
RESULTS AND DICU5SI0N
fnrtial analyses of raw material (PSP and BGR) were to determine moisture, protein, lipid, ash, crude fiber and the anthocyanin contents of the two atwve sutistrates. The results mdk^ated that PSP has an initial moisture content of 64.5%, wtiile BGR of 12%. The protein, fat, cmde ash and fiber content in PSP were 3.89, 0.65, 2.47 and 7.83% (d.b.), reflectively, andttiose for BGR were 7.02, 1.16,1.56 and 4.17% (d.b.), respectively. The protein and fat content in BGR are higher ttian that in PSP, while the fiber content is lower. The anthocyanin content in PSP was 164.69 and higher than that in BGR, whi<^ vras 90.08 (mg Cyd-3-glu-E/ lOOg d.m.).
Reducing sugar of PSP and BGR tiefore and after saccharification, and after fermentation
Total reducing sugar contents of two substtates tiefore, afier saccharification and after fermentation are shown in Rg. 1.
Effects of two e n z ^ e s a-amylase and glucosam^ase on saccharification on PSP were more pronounced than on BGR.
As for PSP substtate, reducing sugar content was raised from 2.54% to 17.85% after 90 minutes of incubation.
Meanwhile, wrth BGR substtate. the increase was from 2.15% to 15.74%. Then, rt was a slightiy reduction in sugar content of BGR after fermentation from 15.74% to 14.58%. Wrth PSP substtBte, reducing sugar content decreased from 17.85% to 15.45%. Other stodies on lactic add fermentation of sweet potato found ttiat redudng sugar reduced appfoximatety 2% after fermenting process (W(»igkhalaung, 1995).
Growth curve of Lacidophilus
After pn^>agation of L. acidophilus in MRS broth, the morphology was checked by Gram staining, under 100X microscopic objective lens. L acidophilus appeared in purple color because of their thick peptidogtycan. They are tiacillus organisms, usually separate from each ottier or form a short chain. This bacterium, is homofermentative and has optimum temperature finm 37-42'C. The agar plates were made writti the sample on ttie surface and spread gradually.
One cell can multiply in geometiic progression to form one colony. After incubating at 37''C in 72 h. the colonies of L.
ackiophilus had w^ite round shape, convex surface vnth a smooth outer edge. Growtti curve of Lacidophilus is established in Rg. 2 in which log (CFU/mL) was recorded every 5 h over a period of 24h.
^- 14 a 12 I- 3 10
^ P S P S B G R
i 9
I;
J s S S i * a 3
I =
S 1
Tin»(h) Figure 1. Reducing sugar {% d.m.) of PSP and BGR solutions Figure 2. Growth curve of L acidopliilus A lag phase extended about 8 h. At this time, the bacteria had to adapt v«th tiie MRS bnath medium. Exponential phase (tog phase) lasted for the next 12 h. PAer that, the growth of bacteria turned to stationary phase. The results showed that the best time to collect starter culture was in range of 10-20 h.
By measunng the OD of broth medium combined with spreading plates, the number of colony fomiing unit (CFU) could t)e measured (Table 1). The starter culture for lactic add fermentation was around 10^ CFU/mL.
HOI NGH! KHOA HQC C O N G NGHf SINH HQC TOAN C - TaMa 1. The correspond number among OD. coti number and Log (CFU/mL) of L acidophilus
OD Cell number Log (CFlWmL)
0.06 - 0.10 2 . 0 1 x 1 0 ^ - 3 . 1 5 x 1 0 ' 3 . 3 0 - 3 . 5 0
0.4 - 0.9 4 31 X 1 0 * - 4 . 0 7 x 1 0 * 4 . 6 3 - 8 . 6 1
1 . 0 1 - 1 . 1 2 4 . 1 6 x 1 0 ' - 5 . 4 0 X l O * 8 6 2 - 8 . 7 3 Lactic acid fennentation of PSP and BGR
Lactic acid fennentation of saccharified PSP and BGR was canied out and total viable cell count acidity and pH changes in ttie fennentation brotti are showed in Table 2. The results showed that PSP was better substrate for L addof^ilus fennentation ttian BGR. Wrth ttie same amount of starter culture, 1% of inoculum 1.02 x 10 (CFU/mL), after 24 h incubation ttie viabte cell count of tactic acid baderia on ttie PSP substt^ate increased to 6.20 x 10 (CFU/mL), as compared to 1.12 x 10^ (CFU/mL) on ttie BGR substrate.
Table 2. Some characteristics of lactic femiented PSP and BGR
Viable cell count (CFU/mL) Acidity {%)
pH
PSP Before fennentation 1.02x10^
0.1 5 1
After femientatwn 6,20x10*
0.54 3.33
BGR Before fermentatkin 1 . 0 2 x 1 0 ' 0.08 5.8
After fermentation 1.12x10*
0.35 3.16
With lactic fermented PSP, pH decreased ft'om 5.1 to 3.33, acidity increased from 0.1% to 0.54% and viable cell count of lactic acid bacteria was raised about 3 tog cycles, from 1.02 x 10^ to 6.20 x 10". Wrth lactic fermented BGR, pH decreased from 5.8 to 3.16, ackjity increased fnm\ 0.08% to 0.35% and viable cell count of lactic acid bacteria was aiso grew about 3 log cydes, from 1.02 X 10^ toi.12x10^
During fennentation, L ackiophSus could piay an important role for fermentation system which ferments monosaccharide. The reduction of pH of fermented PSP and BGR were probably due the formation of acids by bacteria utilizing carbohydrate.
Anthocyanin concentration of PSP and BGR tiefore and after fermentation
Fig. 3 shows ttiat ttie anthocyanin contents of PSP and BGR solution were reduced after fermentation. The anttiocyanin concentration in raw material of PSP (164.69 mg/IOOg d.b.) was higher than that in BGR (90.08 mg/IOOg d.b.). After 24 h of femnentetion, PSP's anthocyanin was decreased from 164.69 mg/IOOg d.b to 102.07 mg/IOOg d.b. BGR's anttiocyanin was also reduced from 90.08 mg/IOOg db to 35.71 mg/100g d.b.
According to ANOVA analysis, ttie anthocyanin concentrations of two substrates, PSP and BGR, reduced significantty after fermentation. Cyanidin-^^glucoside is the most widespread anttiocyanin from fmit v^etebles and plants. Add may cause partial or total hydrolysis of ttie acyl moieties of acylated anttiocyanins (Kong ef al., 2003). In addition, degradation of anttiocyanins In ttie presence of weak adds, consists of direct condensation of add on the carton 4 of ttie anthocyanin molecule (Poei-Langston. Wralstad, 1981). From the results, tiie decrease in total monomeric anttiocyanin content resulted from pH lowering during fermentation
DPPH radical scavenging activity of PSP and BGR before and after fermentation
Bafbra femwnt.
Figure 4. DPPH radical scavenging activity of the PSP and BGR solutions
Results of ttie tree radica scavenging caDacih are nrp-iflntori in Pin A T-.KI= ^ t. --uuno iiiyjauiiny or dnirgxiuaiiu..
oe i r .,->!,.« rth= ^^r,^»-t«.- . 1? T^^"-^* ^'^ presentea in Pig. 4. Table 3 shows the antioxidant activtv evauated
as ICso value (ttie concentration at which radical scavenging activity is 50%). «vaiua»
° ' ; C O N G N G H E S I N H H O C T O A N Q U O C 2 0 1 3
R g . 4 m d k a t e s that for b o t t i t t i e P S P and B G R solutions ttie D P P H radnal s c a v e n g i n g activity d e c r e a s e d signrtrcantty after f e r m e n t a t i o n . In c o r r e l a l t o n t o ttie a n t t i o c y s i i n c o n t e n t t h e antioxidant activity of t h e PSP solutions w a s higher than t h a t o f ttie B G R s o t u t k x i s .
T h e a n t i o x i d a n t potential is inversely proportional to ICso v a l u e . It m e a n s ttiat ttie higher t h e IC50 v a l u e s w e r e , ttie lower ttie a n t i o x i d a n t activrties w e r e . According to Tat>le 3, P S P solution before femientation s h o w e d strongest antioxidant activity (IC50 = 1 8 . 6 1 % ) . A f t e r femientation b y L acKtop/rfus. I C s i v ^ u e s of P S P a n d BGR increased f r o m 1 8 . 6 1 % to 3 1 . 1 9 % a n d f r o m 3 7 . 1 9 % to 9 6 7 0 % , respectively. D u e to ttiis r e s u l t P S P solufion exhibrts more antioxidant activity ttian P S P s o l u t i t x i , tx)th t w f o r e a n d after fermentation. T h e free radical scavenging activrty in tiie f e r m e n t e d solution is attributed to ttie a n t t i o c y a n i n c o m p o u n d s in P S P and B G R . Therefore, ttie antioxidant activities of t w o substrates w e r e r e d u c e d d u e to t h e r e d u c t i o n i n anttiocyanin contents o f both P S P a n d B G R after fermentation.
Talile 3. I C a value of t h e PSP and BGR solirtians
PSP
BGR
Before fermentation A f l ^ femientation Before fenn^itation After fernierdutmn
ICa (%, vN) 18.61 31.19 37.19 96.70 C O N C L U S I O N
Saccharfficati(»i o f P S P a n d B G R witii t h e c o m b i n a t i o n o f 0 . 1 % o - a m y l a s e and 0 . 1 5 % glucoamylase i n 9 0 m i n , a t 5 5 ° C produced solutions wrth t h e r e d u d n g sugar content surtable fiar ttie femientation with L. addophillus. T h e saccharified PSP solution c o n t a i n e d h i g h e r level o f reducing s u g a r artd a n t h o c y a n i n c o m p o u n d s t h a n ttie B G R solution. By using 1 % starter CLrture of L addof^ilus, falcultative anaerobic fermentation o f t w o sutistrates w e r e carried o u t for 2 4 hours a t 37°C. Lactic a d d f e r m e n t a t i o n increased a d d i t y , reduced p H v a l u e , anthocyanin content and antioxidant capacity of t h e fermented s d u t i o n s .
B a s e d o n t h e viable c e l l c o u n t , this study s h o w e d that PSP a n d B G R were t h e appropriate substrates for lactic acid fermentation of Laadophllus. Therefore, it c a n b e c o n d u d e d that there were virtuous possibilities to develop products o f tieverage with antioxidant activity f r o m lactic fermentation of saccharified PSP and B G R .
R E F E R E N C E S
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Duangjitctiaroen Y. Kantachot D, Ongsakul M, Poosaran N, & Cti^yasut C (2008) Selection of probiotic lactic bacteria isolated from fermented plant beverages. Pafc J SKI/Sc), 11(4): 652-655.
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HQI NGH! KHOA HOC CONG NGHE SINH HOC TOAH
L E N M E N LACTIC TLf KHOAI LANG TIM VA GAO N t P THAN VCXI C H O N G A LACTOBACILLUS ACIDOPHILUS VA K H A O SAT KHA NANG C H O N G OXY HOA^
TR^N DUNG DICH SAU L^N MEN ^
D$ng Qu6c Tuin *, Ly Hong Van Nhi
Tnjong Dai hgc <3u6c ti - Dgi hoc Quoc gia Tp. Hd Chl Mmh
T6M TAT '\
Cong trinh n ^ e n oiu n ^ n b ^ tim la tii£n vpng len men latic tren bai co chdt kboai lang tim {Ipomoea batatas L) va gao n ^ flui {Oryza sativa L) vdi chijii^ Lactobacillus aadopiulus, ciing nhu khao sal kha nang ch6ng oxy boi vi him lugng anthocyanin c ^ dung dicb sau len men. Khoai lang tim (I^P) va g^o oep tiian (BGR) duoc ducmg b6a boi sv iKt hftp cua nhiing loai amylolytic enzyme: 0,]%a-aniilazavi 0,15% ghicoamilaza. Dung dich dinmg b6a khoai lang tim va gao nqi than, co ham l u ^ e dufmg Idni 1 ^ ]ir?t Ii 17,85 vi 15,75% (tinh tbeo cbit kho), du^c dung lim co chit cbo qui trinh len men lactic v6i 1% ti 1? gifing vi kfau^^
acidophilus ban diu. Len men kj kfii khong bit bu$c duyc din banh trong v6ng 24 gioi dnhi^ d$ 37°C. Dui^ dich khoai lang ttia a ^ len men chiia 6,20 x 10* CFU/mL mSt d$ vi khuin vi d6i vdi dung djcb g ^ n ^ dian sau \ea men li 1,12 x lO' CFU/mL. Kim t n ^ anthoc^nin tnmg djch PSP giam ding ke sau len men, tii 164,69 xuong 102,07 mg Cyd-3-glu-E /lOOg (chit kho). Xu htidiign^
ciing diiy a djch BGR, bim hipng antiiocyanin gjam tii 90,08 xu6ng 35,71 mg Cyd-3-glu-E /lOOg (chit kho). Khi ning c h ^ o | h6a ciia dung djch tmoc vi sau l£n men dugc danh gii bing phuong p h ^ quet goc tu do l,l-Diphciiyl-2-picr^ydrazyl (DPPI^n v^y, CO th^ dua ra de nghj ting san phim len men lactic tii khoai lang tim vi gfio nep than co (he dupe su dyng de phit trice ~'- phim vi thiic u6ng dinh dudng vdi svr tid sung cua vi khuin c6 Igi vi kha nang chong o-xy boi.
Til khda: khoai loang tim, gao n ^ ihan, Lactobacillus acidophilus, anthocyanins, kha ning chSng o-xy hoa
' Author for correspondence. Tel: (84-8)37244270 (ext. 3824); Email: dgluanjajhcrniu.edu vn
