HOI NGHI KHOA HQC C O N G NGH? SINH H p C T O A N Q U O C 2013
IMPROVEMENT OF SOLUBLE SOLIDS CONTENT OF COFFEE FERMENTATION BY USING BIO-PRODUCTS FROM ASPERGILLUS
CARBONARIUS, PICHIA GUILUERMONDII, AND BACILLUS MEGATERIUM
Nguyen Duy Trung', Le T h l Hong Tuyet^, Le H o n g P h u ' '
^Intemabonal University, Vietnam NaSonal UniversHy, Ho Chl hAinh City
^University of Natural Resources and Environment, Ho Chl Minh City
SUMMARV
The soluble solids extraction &om coffee can be widely done by chemical and physical nudiods but this study focused on biochemical method. The soluble solids extraction (SSE) is nomially limited by two major protective components of coffee bean, cellulose and pectin. Io fiiis research, biocoffee-7, the bioproduct synthesized fiom a specific microorganism, by the culture containing rice bran, rice husl: and canot (produced from Aspergillus carbonarius which had h i ^ enzymatic activities (cellulase |nd pectinase), was combined mdi biocoffee-12 (produced from Pichia guilliermondii) or with biocoffee-17 (produced from Bacillus megatenum) for coffee fermentation. Soluble solids content was successfiiliy increased 68% in w4ucb caffeine content increased 46%
in comparison with control samples. Furthermore, sensory evaluation proved ttiat the coffee products had good coffee state, color, aroma and taste. '
~ Keywords: Aspergilius, bio-product, coffee, fermentation, soluble solids INTRODUCTION
Inslanl coffee, also called soluble coffee and coffee powder was invented in 1901 by Salon Kalo, a Japanese scientist working in Chicago. Inslanl coffee is commercially prepared by either freeze-drying o r spray drying. Advantage o f instant coffee is its simplicity o f preparation. Yield and^quality of instant coffee are both depended on soluble solids content gained ^ m a process called coffee extraction.
Coffee product, produced by traditional method (dry method) has a large amount of soluble solids still left in t h e coffee- grounds after extraction. The resistance of coffee extraction i s mainly due to two components, pectin and cellulose in the coffee bean (Luong N. D. 2008). These components have stable biological structures and are main Actors in protective layers orcoffee beans.
In this research biocoffee-7 (produced f i p m Aspergillus carbonarius), biocoffee-12 p r o d u c e d from Pichia guliiermondii) and biocoffee-17 (produced ftom 6ac///us megaterrum) were used to ferment coffee beans. These microbial species have high pectinase and cellulose activities. When they are cultured in the solid state and liquid state (R Kavitha, S Umesh- Kumar, 2000). The enzymatic hydnslysis of pectinolytic and cellulolyllc substances b y microorganisijis resulted in, the higher soluble solids content o f extraction in fermented coffee comparing l o unbBated c o n & l . In addition P.
guilliermondii and B. megaterium have been widely used in food technology to increase flavor of foods (Andre, Barata, 2006). The objectave of this research was to Improve soluble solids content ( t h e enhancement soluble solid content was - processed by breaking down the cellulose and pectin by fermenting the beans fi-om microbes as A. carbonarius, P.
guliiermondii and B. megaterium try coffee fermentation in which bio-produdsjvere combined t o g e ^ e r . The research contents included (1) optimizing the femientation cbnditions such as moisture content, ratio o f bio-products a n d coffee beans and fermentation duration and delemiine the pH changes as well. (2) C^nying out coffee fermentation i n w^ich combined biocoffee-7 with biocoffee-12 or vAth biocoffee-17 for fermentation. (3) Testing toxiaty and determining caffeine content of fermented coffee, (4) C a r r ^ n g out sensory evaluation of fermented coffee.
MATERIALS A N D METHODS Materials
Coffea robusta was taken from Daklak provincewhere this kind of coffee is mosfly produced in Welnam.
Aspergillus carbonarius, P. guilliennondii and e . megaterium were pnavided by Laboratory of Cell Biotechnology, Room 701, International University HCMC.
Blo-products
Biocoffee-12 and biocoffee-17 were produced from P. guilliermondii and B. megaterium when the miSroorganlsms cultured in suitable media. Biocoffee-12 contained 1.8 x 10^ CFU/ml and Biocoffee-17 contained 2,5 x 10^ CFU/ml.
Table 1 . T h e composiSons o f media were used In p r o d u c i n g biocoffee-12 a n d biocoffee-17 ComposiSons
Name of bitHHoduds Cafrol Time pH (NH,)jSO, {%) T" fC) "Brix powder (hours)
(%>
HOI NGH! KHOA HQC CONG NGHE SINH HOC TOAN QUOC 2013
_BJQf^ffep-7_w3s pmriiirjqd-fmm A. carbonarius when the microorganism cultured in medium with compositions: canot powder 6%. rice husk 18%, rice bran 7 5 % and moisture content 56%. After culturing for 42 hours at the room temperature, the medium was mixed vnth rice powder at ratio of 1:1 and then ground well, stored in cool fridge.Biocoffee- 7 stored for 90 days, the number of cell deducted over the time and about 32% at 90 days, however; Biocoffee-iz s Biocoffee-17 had lo use after production.
Methods ' . • -~ - ' , . . . , Detennination of the cellulose content by Soxhiet extractor (Mui N. V., 2001), DetemlnaUon of pectin content by pedate calcium method (Can-e and Haynes, 1922), Determination of soluble solids content by Refractomeler machine Fermented coffee beans were put into electric drying oven at 60° C and drying time was 24 hours. After drying, the coffee beans were roasted by coffee roaster at 250° C and in 12 minutes. Before coffee extraction roasted coffee beans were ground well by coffee grinder. In order to extract coffee, 5g of coffee powder was put into filter vnth 10ml boiling water then coffee solution was used to calculate soluble solids content by Keffactometer machine.
Experimental design
Coffee femientation using biocoffee-7, 100 g of coffee contained in ffiin towel was soaked in distilled water in different durations: 0,5, 1 , 1.5, 2, 2.5, 3, 3.5. 4 and 4.5 hours relatively the moisture content was reached 28.B5, 33.55, 40.38, 45.15,47.20,49.67,51.85, 53.57. and 53.79% respectively. After soaking the towels contained coffee put on baskets for few minutes to be partially drainefl. After that biocoffee-7 was put into the lOOg of coffee vntii different ratios of blocoffee- , 7 and coffee were: 1,2, 3, 4 and 5%, mixed them well and used the towels to cover coffee mixed biocoffee-7. At last, the masses of fermented coffee placed dry and clean area at room temperature in different limes. 1 0 , 1 5 , 20, 25. 3 0 , 3 5 , 4 0 hours. Noting control samples were not mixed witii biocoffee-7.
Coffee femientation using biocoffee-12_ and biocoffee-17, lOOg of coffee put into plastic Jar after that biocoffee-12 or biocoffee-17 vJas poured into the jar with different ratios of bio-pnsducl and coffee: 10, 20, 30, 40, 50 and 6 0 % . Then distilled water added until reached 300ml. Finally, the jars put on shaker at velocity of 11Srpm and kept at different times:
10,15, 20, 25, 3 0 . 3 5 , 4 0 hours. -
Ferrnentallon combined biocoffee-7 with biocoffee-12 and combined biocoffee-7 with biocoffee-17, 100g of coffee fermented by biocoffee-7 at optimum conditions was washed and then fermented by biocoffee-12 or biocoffee-17 at optimum conditions.
Testing iflatoxin and determining ^caffeine content of femiented coffee at Quality Assurance & Testing Center 3 (49 Pasteur, district 1, H o ' C h i Minh City, Vietnam) with AOAC 2007 (991.31) and AOAC 2007 (979.08) methods.
respectively. J RESULTS A N D DISCUSSION
Cellulose and pecliii are polysacchandes found in coffee with percentages of 3.6 and 28.8% r e s p ^ v e l y . ~ T h e s e were major components of coffee which Inhibited soluble solids extraction since they are very hard protective layers of coffee.
Aspergillus carbonarius produces two enzymes cellulase and pectinase which hydrolyze cellulose and pectin into single sugars and used as source of carbon for microorganism's growth.
Optimum moisture content of coffee f o r fermentation
Moisture content is a fector significantly impacting on the microbial
•growth; especially fungi Since" water 1s'""£r'solvent for 'most o f ' biochemical reactions inside cells, bul Uie cells often need a certain amount of water.
In addition, hydmlysis of cellulose and pectin by cellulase o r . pectinase respectively also needs water.
Waler is absorbed into celLfrom_outsjde_environment by osmotic pressure. Therefore, the survey of coffee moisture is essential to collect desired enzyme amount, and lo hydrolyze cellulose and pectin better. ^^.-
Flrure 1. the change of SS content extracted from coffee fermented by coffee-7 at different moisture content
In order to survey optimum moisture content of coffee beans, e a d i lOOg sample was soaked in water with individual time to get different moisture contents as method presented above. The lOOg samples were continuously mixed with the -same amount of biocoffee-7 at 56% (4 g) and fermented with the same time (30 hours).
Figure 1 is shown that at moisture content of 40.39% soluble solids content extracted highest (3.74°brix} it means Uie optimum moisture content of coffee is 40.39%. At the moisture content, microorganisms grew rapidly and produced high amount of desired enzymes. Moreover, pectinase & cellulase are hydrolytic enzymes, so Uiey also worked with wafer.
Determining the optimum moisture content of coffee for femientation only cam'ed aut when coffee fermented by biocoffee-7. For biocoffe6-12 or biocoffe#-17 used to ferment coffee at liquid stage therefore no need to determine.
HOl NGH! KHOA HQC C O N G NGHE SINH HQC T O A N Q U 6 C 2013
D e t e r m i n i n g the o p t i m u m ratio o f b i o - p r o d u c t s a n d coffee beans f o r coffee fermentation
In order lo detemnine optimum ratio of biocoffee-7 and coffee beans 100 g coffee samples were soaked in the same time so they got the same moisture content (40.39%). The coffee samples were mixed with different amounts of biocoffee-7 and fermented in ttie same fermented duration (30 hours).
In the cases o f biacoffee-12 and 17, 100 g coffee samples were also mixed witii different ratios and fermented with the same fermented duration (30 hours).
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cg|leabeini(W - > - RatoolbliiciitTet-lia
RaliD of biacDffee-7t coffee beans (11 R«lnntiHnRnfliinAcnf(iiiihiwKiv,i Figure 2. The change of SS content extracted from coffee Figure 3. The change of SS content extracted from coffee fermented by biocoffee-7 at different ratios fermented by blocoffee-12 and biocoffee-17 at different ratios The figure 2 is shown that soluble solids contents extracted writh the highest amount 8.56 (" brlx) as the ratio of biocoffee- 7 put into at the beginning of fermentation was 4 % . Soluble solids content increased at the ratios of 1 % to 3 % , and reached maximum at ratio of 3% w/w after thai soluble solids contents rapidly decreased a l the ratios of 4 % to 7 % . , The figure 3 is shown that the optimum ratios of biocoffeB-12 and biocoffee-17were40%v/w. The figure S-is also shown that SS contents extracted when brocoffee-17 used for coffee fermentation was always higher than SS content extracted when biocoffee-12 used for coffee fermentation a l all ratios respecb'vely. Biocoffee-12, Biocoffee-17 containing living microbes so they could use soluble solids if time was long enough or ratio was high enough.
Determining the o p t i m u m f e m i e n t e d d u r a t i o n f o r coffee f e r m e n t a t i o n by hlo-prodUcts
To determine optimum fermented duration for biocoffee-7, the lOOg-coffee samples were mixed with the same ratio of biocoffee-7 and coffee beans (3%) and soaked in water with the same time to get same moisture content (40.39%) and then the samples were fennenled with different durations.
To delemiine optimum fermented duration for bioooffee-12 and 17, lOOg-coffee samples were mixed with tiie same ratios of bio-products and coffee beans (40%) and fermented with different durations.
^ - r r j - ^ ^ ' 4/6333
^n 1667
CT 10 15 20 25 30 35 49 Femiensd duraHona fltoursr
m
1
10
s s 7
5
r'
cr 10
-o-Colliihinen^ciiiti • blocoffj^iz
15 Zli 25 30 35 il Fernienud duratfons (hours]
Firgure 4. The change of SS content extracted from coffee Firgure 5. The change of SS content extracted from coffee fermented by biocoffee-7 at different durations fermented by bfocoffee-12 and biocoffee-17 at different
durations
The figure4 is shown that soluble solids extracted are increased from 10 to 30 hours, and soluble solids content reaches maximum at duration of 30 hours. It means thai the microorganism {A. cariionarius) grew up and the number o f cells increased gradually and they produced high amount of enzymes (cellulase. pectinase) which hydrolyzed cellulose and pectin compounds. In this case, the soluble solids were easily extracted.
In oUier hand, the figure 4 showed that soluble solids content increased gradually from fennenled durations of 3 5 to 55 .hours. Because the number of cells increased rapidly in the times, it also means the enzymes were produced higher. But Ihe cells also needed large amount of nutrient for tfieir growth and they used soluble solids as their foods which explained why amount of soluble solids decreased rapidly.
In conclusion, the optimum fermented duration for fermentation using'biocoffee-7 was 30 hours. When coffee fermented by biocoffee-12, SS contents extracted was increasing from 10 hours to 25 hours. The highest SS content achieved at 25
HOnrdHTKROAT
hours, and if the femiented durations were over 25 hours the SS contents received fall down. In condusion, the optimum
fermented duration for biocoffee-12 was 25 hours, (l-igure b) ' ' ~ The highest SS content 10.18 f brix) created when Uie coffee was fennemed by biocoffee-17 in 30 hours. If fermented
durations were over 30 hours SS content decreased. (Rgure 5) Combined fermentations , , . . . _ . Table 2. SS content extracted from combined coffee fermentatian
12.58 11.81
67.70 57.50
CT control sample. AB coffee fermentation combined biocoffee-7 and biacoffee-17. AP coffee fermentation combined bioco(fee-7 and biocoffee- 12. PB coffee fennenlalion combirfed biocoffee-12 and biocoffee-17; APB coffee fermentation combined biocoffee-7,12 and 17
PB 7.88 5.10 According to (table 2) SS content extracted ABP . 7.60 1.30 was increasing significanUy when coffee
1 fermentation combined biocoffee-7 with biocoffee-12 or witti biocoffee-17. The SS content extracted increased from 57.5 lo 67.7 % respectively In comparison to control sample (coffee was not fermented). Other combinati'ons did not have desired results. Evenly SS content extracted decreased In comparison to samples in which coffee fermented by each bio-products. The factors for increasing the SS content were both lime fermentation and also enzymatic of microorganism.
The change o f pH d u r i n g coffee fermentation at o p t i m u m c o n d i t i o n s
The Fig. 6 showed ffie pH was be changed from pH 6 to pH 3.75 during with blocoffee 17 and 12 fermentation. The reason might be fermentation they transformed some compounds in the media Into acids
Quality of fermented coffee
Aflatoxins commonly found in fungal species, are toxic for human. They cause symptoms .such as vomitting, abdominal pain, pulmonary edema, convulsions, coma,
^and death with cerebral edema and fatty involvement of the liver, kidney, and heart AB and AP were two best products chosen to test aflatoxins at Quality Assurance
& Testing Center 3. The lest showed tiiat there were not anatoxins' found in the products. That meant ftie
products were safe to human.
Caffeine also tested al Quality Assurance & Testing Center 3, Uie lest showed that amount of caffeine increased in comparison lo control sample. In the product AP and A B caffeine content increased 12.6% and 4 6 % , respectively .Thai also affirmed soluble solids content increased in two fermented coffee products AP and AB.
/^cording to sensory evaluation canied oul by specialists, the products AP and AB were two good products with mean markswere 1 6 , 4 5 a n d i 7 . 2 2 respectively ' • ' • - • ' • ' - " " - . - • • " - -- -^ .... — - - • - - : — - • In summary, determining the optimum conditions of coffee fermentation in which three kinds of bio-products were used _ (biocoffee-7, biocoffee-12, biocoffee-17). The optimum conditions detennined for each kind of bio-products shown in (table 3),
Table 3. Optimum conditions for blo-producta used In coffee fermentation B!o-products
Optimum conditions^
Optimum moisture content (%) Optimum ratios (%) Optimum fermented duraliona
SS 5 4.6 4 3,S 3
i ^ H ; ^ ^
—B- biocffee-l?
—"- Biocoffee-ia
"%«%
Y**^
4 J 6 .425 ^ ^ 43• - ^ 8 3 7
CONCLUSION
While the research carried out by BSc Cam Van (2010), coffee fennenled by Aspergillus niger soluble solids content only increased 28.6%. This research also succeeded In increasing significantly soluble solids content in two products (AP and AB). The soluble solids content increased fnam 57.5% and 67.7% respectively, caffeine content increased 4 6 % - compared with control samples. Furtiiermore AP and AB products tested were safe to human, and according lo sensory- evaluation showed tiiat the two coffee products had good quality.
HQI NGH! KHOA HQC C O N G NGHE SINH HQC T O A N Q U O C 2013
REFERENCES
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Chapin III, F.S, P A Malson, H A Mooney, (2002)." Principles of Terrestrial Ecosystem Ecology. Springer-Veriag New York, NY Kavitha R, S Umesh-Kumar, (2000). Potential application of pectinase In developing functional foods, PubMed Nhan D. T., (2001). Intemationel Coffee Conference
Nkondjock, Andr6, (2009) Coffee consumption and the nsk of cancer An overview, p. 121-125 .- Noah Daniels, (2009). Variations in Coffee Processing and Their Impact c»i Quality and Consistency Mui N v., (2001). Lab On &odierdstry
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NGHIEN CLTU NANG CAO CHAT TRICH RAN H 6 A TAN TO" CA PHS BO^I LEN MEN K£T HOP CAC C H £ PHAM TO" ASPERGILLUS CARBONARIUS, PICHIA GUILUERMONDII VA BACILLUS MEGATERIUM
Nguyen DuyTrung^, L e T h i Hong Tuyet', Le Hong PhO^"
'Tnrbng B^i hgc Quoc te, Dd hoc Qu6c gia Tp Ho Chi Minh Trubng Dd hgc Tki nguyen vi Moi trubng Tp Ho Chl Minh
T6MTAT
Cic chit ran hfia tan chiSt xuit tii cA phfi c6Ak _du^ thvc h i ^ rOug rSi bSng cic phucmg pliap h6a hpc va v^t Iy nhimg nghien c&u niy t|p trang vio piiuong phap sinh hoa. Cic clilt rSn h6a tan chi^t xuit diuong dugc gi6i han bdi hai thinh phin bio v? chii yeu ciia ci phe, cellulose va pecdn. Trong ngliifin ciru niy, biocoffee-7 (sin xuit tur Aspergillus carbonarius c6 cac hoat tinh enzyme cao (cellulase v i peclmase), d u ^ kll hop voi biocofFee-12 (dugc sin jcult t i Pichia guilliermondif) hoac v6i biocoffee-17 (duoc sin xuat tii vi khuan 5aci//uj megaterium) Irong qui trinh len men c i phe. Him lugng chit rin hda tan dugrc tang ten 68%, trong d6 ham iugng caffeine tang 46% so v6i mlu ddi chfrng. Hon niia, danh gia cim quan chiing minh cac sin phim ci pbe c6 cim quan tot nhu miu sac, nuii thom v i huong vj.
J^r khda Aspergillus, che phim, ca phe, len men, chit tin h6a tan
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