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Imicrobial kinetics, fermentative and chemic

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Vol 82. No 10 (2012) Microbial kinetics,fermentative andchemical characteristicsin solid statefermentation of applebagasse

,\I1SI R,\('J 1'1)1'

y CASTILLO-CAST/LLO, O RU/Z-BARRERA, J SALlNAS-G/AV/RA, C ANGULO-MONTOYA, E BURROLA-BAIIRAZA, M MUR/LLO-ORTlZ M MONTA ÑO-G, A ELÍAS-IGLES/AS

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26/11/2012

Indían Journal of Animal Sciences 82 (lO): 9~O, October 2012

Microbial kinetics, fermentative and chemical characteristics in solid statefermentation of apple bagasse

y CASTlLLO-CASTILLOl, O RUIZ-BARRERA2, J SALlNAS-CHAVIRAJt, C ANGULO-MONTOYA4,

E BURROLA-BARRAZA5, M MURILLO-ORTIZ6, M MONTAÑO-G7 and A ELÍAS-IGLESIAS8

Facultad de Zootecnia y Ecologia, Universidad Autánoma de Chihuahua, México

Received: 28 December 2011; Accepted: 25 February 2012

ABSTRACT

Thc objective of this study was to evaluate the microbial kinetics, ferrnentative and chemical characteristics duringsolid state fermentalion (SSF) of apple bagasse (AB), which was determined over the course of 4 incubation times; O,24, 48, 72 h, in a completely randomized design with 4 repetitions. pH values, lactic acid concentration, numbers oftotal aerobic bacteria, yeast and lactobacilli, dry rnatter digestibility and neutral detergent fiber digestíbility weredetermined. True and crudc protejo, neutral and acid detergcnt fiber were also measured. Results revcaled that pHdecreased over the 4 sampling times. The lactic acid concentration increased over time. There was a reduction innumbers oftotal aerobic bacteria. Numbers of lactobacilli also reduced. Yeast populations (CFUlml) were stable at 24h, but decreased thereafrer. In vítro dry matter digestibility (lVDMD) increased during incubation. In vitro neutraldetergent fiber digestibility (IVNDFD) similarly increased, with a máximum value observed at 72 h. True protein (TP)increased during fermentation, achieving a high value at 24 h: howcver, crude protein (CP) showed no change duringincubation. Neutral detergcnt fiber (NDF) content did not change during fermentation however acid detergent fiber(ADF) reduced. It is concluded that the increased content of lactic acid and the accompanyíng dccrease in pH duringSSF of AB negatively affecrcd the yeast and total bacteria populations whereas (me protein coment increased likelybecause of formation of unicellular protein during the process,

Key words: Apple waste, Bacteria, Chemieal eomposition, Fermentation, Yeast

Currently, biotechnological methods sueh as solid statefermentation (SSF) of agro-industrial byproducts to enriehPresent address: tycastiIl075@yahoo.com Departamento deMedicina Veterinaria, División Multidisciplinaria, UniversidadAutónoma de Cd, Juárez, Avenida Universidad, Sección Hidalgo,31333 Nuevo Casas Grandes, Chihuahua, México;2oscaruiz@uach.mx Facultad de Zootecnia y Ecología, UniversidadAutónoma de Chihuahua. Km 1 Periferico Feo. R. Almada, C. P.31031 Chihuahua, Chih., México; ljsalinasc@hotmnil.comFacultad de Medicina Veterinaria y Zootecnia, UniversidadAutónoma de Tamaulipas. Matamoros 8 y 9 Z. Centro. C.P. 87100Cd. Victoria. Tamps. México; 4c.angulom@hotmail.com CarroIntem, Km 3.5 Sur, Culiacán, Sinaloa, México; sburrolav@uach.mxFacultad de Zootecnia y Ecologia, Universidad Autónoma deChihuahua, Km 1PerifericoFco. R.Almada, C.P. 31031 Chihuahua,Chih .. Mexico; 6muom58@hotmail.com Facultad de MedicinaVeterinaria)' Zootecnia, Universidad Juárez del Estado de Durango,Carro Durango-Mezquital C P: 34162 Durango, Dgo., México;7mmontano5@yahoo.com Instituto de Investigaciones en CienciasVeterinarias, UABC. Obrcgón y Julián Carrillo, Col. Nueva,Mexicali, B.C. 21100 México; 'aelias@ica.co.cu Departamento deCiencias Biofisiologicas, Instituto de Ciencia Animal, Ministeriode Educación Superior Carr. Central Km 47.5 Apdo P. 24Mayabeque, Habana, Cuba.

protein contents for animal feediug are importanl beeause ofthe low cost and easy operation. Apple bagasse (AE) is themost abundant byproduet produced during juice extraetion.This residue is rích in soluble carbohydrates and pectin(Vicente et al. 2005), and microorganisms like yeasts(Martorell, 2006) which are low in protein but of highbiological value (Rodríguez et al. 2008). During SSF themicroorganisms in the substrate use nitrogen, carbohydrates,minerals and vitamins for their growth and reproduction.Conscquently, the final produet is enriehed in high qualityprotein and low fiber contents (Rodríguez et al. 2006). Forinstance, Rodríguez et al. (2001) reported an inerease in trueprotcin in SSF of sugar cane (Saccharum officinarum) andswcet polato (Ipomea balata) due lo propagatiou of itscpiphyte flora. Nevertheless, the researeh using AB assubstrare for SSF is limited. The present research objeetivewas to determine fermentation and ehemieal eharaetcristiesand assess mierobial work during SSF ofapple bagasse undermicroaerobie condition.

MATER1ALS AND METHODS

Location: The present researeh was pcrformed in the

October 2012J SOLlD STATE FERMENTATION OF APPLE BAGASSE 97

Animal Nutrition laboratory of the Paculty of AnimalHusbandry and Ecology at the Autonomous University ofChihuahua located in Chihuahua, México.

Experimental procedure: Apple bagasse (AS) obtained.ground and rnixed (Elias el al. 1990) with 1.5% urea, 0.2%ammonium sulphate, and 0.5% a mineral salts mixtureeootaining maero and micro elernents. These ingredients weremixed and then 342 g portions were distributed to sepáratesterile 500 mi ErJenmeyer f1asks for solid sta le fermentation(SSP). Each flask was plugged with cotton and incubatedunder static conditions at 32°C for O, 24, 48, and 72 h. Ateach sampling time, 4 flasks were withdrawn from theineubalor and the total eontents of eaeh flask were eolleetedand individually hornogenized. A 5 g sample of eaeh resultanthornogenate was eombined with 45 ml sterile distilled waterin a 100 mi Erlenmeyer flask and shaken for 30 min withshaker equipment and then filtered through sterile gauzesand refrigerated at 4° C for further analyscs.

Laboratory analysis: The pH of the filtrate obtained asdeseribed in the experimental procedure, was immediatelyreeorded with a table potentiorneter. For quantifieation oflactic acid, 0.25 mi from the sample filtrated was dilutcd in9.75 mi of distilled water. Frorn this solution, 0.5 mi wasused for lactic acid determination aecording to theealorimetric method described by Madrid el al. (1999). Themicroorganism extractions wcre made with the sameprocedure used for aqueous exlractions for pH mcasurcmcnt,but in aseptic conditions. An aliquot of 1 mI of filteredsuspensión was serially dilured to 1O-1J in peptone-water(0.01 'lo). Dilutcd samples were sprcad lo plate eount agar,malt extraer agar supplemented with 0.01 g chloramphenieoVL, and to MRS agar, for enumeration oftotal aerobic bacteria,

yeast and laetobaeilli, respeetively. All ineubations used 4dilutions; for total viable bacteria wcre lO' to ]06, for yeast10' to 107 and lactobacilli 105 to lOS. ínoculated media wereincubated al 37°C aerobically for 24 h to enumerate totalaerobes and yeast and anaerobieally for 48 h to enurneratelaetobacilli. These were earried out in a count-coloniesehamber. The ehosen dilutions that had 30 to 300 colonieswcre eounted. Dry matter (DM), organic matter (OM), ashes(AS), and crude protein were analyzed according to AOAC(1990). For true protein (TP) the method ofBerstein (1970)was used as per Meir (1986). Neutral delergent fiber (NDF)and acid detergent fiber (ADF) were rneasured aecording toVan Soest (1994). These analyses were sequentiallydeterrnined in the fiber analyzer , using filter bags F57 witha porosity of 30 microns. In vítro dry rnatter digestibility(IVDMD) and in vitro neutral detergenl tiber digestibility(IVNDFD) values were obtaincd with incubator (ANKOMTeehnology 1998).

Statistics: A cornpletely randomized design was used with4 repetitions by eaeh sampling time. The slatistieal analysisofthe data was made using the GLM procedure (SAS, version9).

RESULTS AND DlSCUSSION

Fermentation, chemical characteristics and microbialgrowth: The results are shown in Table l.

The pH dccreased signifieantly (P< 0.007) at 24 h andthen rcmained almost unchanged. The results on laetie aeidconcentration through the 4 fermentation times indieatedincrease until 72 h which may be related to the growth oflaetic aeid bacteria. During the fermentation processammonia nitrogen eoneentration increased (P< 0.0009) as

Table 1. Fermenrative, chemical characteristics and microbial growth of the apple bagasse under solid state fermentation

Itern (%) Fennentation times (h) SEM p-

O 24 48 72

Ph 4.13' 3.85b 3.86b 3.81b 0.05 0.007N-NH, (mmol/g of DM 1.92' 1.95' 1.99b 2.00b 0.01 0.0009Lactic acid (ug/g of DM) 22.24' 22.50' 22.87b 23.75' 0.13 0.0001Total bacteria' (CFU/ mi) 7.14"(1.4xI0') 5.96b(92xI0') 4. 73'(5.4x 10') 4.55"(3.5x 1al) 0.03 0.0001Lactobacilli! (CFU/ mi) 8.60'(3.9xI07) 8.14h(1.3xI07) 8.73'(5.4xI07) 8.53'(3.4xI07) 0.02 0.0001Yeasi ' (CFUI mi) 7.42'(2.6x 106) 7.35'(2.2x 106) 6.0b(lxI0') 4.85'(7. l x 1O]) 0.05 0.0001IVDMD% 89.0' 89.21' 92.IOb 94.28' 0.67 0.0005

IVNDFD% 81.1' 83.41' 87.11 b 88.63b 1.29 0.004

Dry matter 18.0' 18.92b 18.97b 19.12b 0.16 0.001

Organic matter 95.86' 96.14b 96.13b 96.01b 0.07 0.05Ashes 4.14' 3.86b 3.87b 3.99b 0.07 0.05

True protein 16.89' 21.15b 19.50·b 18.12'b 0.91 0.03

Crude protein 36.19 36.02 35.57 35.47 0.30 N.S

Neutral detergent fiber 52.65 53.14 53.22 53.76 0.45 N.S

Acid detergent fibcr 41.57' 41.45' 38.80b 39.42b 0.30 0.0001

• b e d Dífferent superscripts in the same row difTcr statistically; I Thc counts of microorganisms wcre according lo Iogarithmic function.CFU,colony-fonning unit; IVDMD, in vitro dry rnatrer digeslibilily;IVNDFD, in vitro neutral derergent fiber digestibility.

GJ

98 CASTILLO-CASTILLO ET AL. [Indian Joumal o/ Animal Sciences 82 (10)

the incubation time progresscd (Table 1). This could berelated to the ureolytic action of urease-positive bacteria thatnormally occur in SSP processes (Valiño el al. 1994).Moreover, protein synthesis takes place more slowly thanurea hydrolysis (Rodriguez el al. 2001). The air present inthe SSF mixtures limited the growth of lactic bacteria at 24h; but al 48 h their growth was favored by oxygenconsumption and pH reduction (P< 0.0001). Acid tolerantLactobacilli are able to grow at pH of 3 or below (Hyden2001, Pandey el al. 2001). The de crease ofLactobacilli at72 h was attributable to substrate decline, which also causeda reduction in total bacteria and yeast counts. The results ontotal counts of aerobic bacteria showcd a decrcase (P<0.000 1) as the fermentatioo time passed. This dcpressiveeffect on total bacteria population could be rclated to thehigh lactic-acid concentration, which influences on pHreduction causing a bactericidal effecl (Hyden 2001). In thefirst 24 h the yeast population was constant, during themaintenance phase, and then it came into mortal phase, whichcould be attributed to low pH of the ecosystem, which islethal for yeast (Elias and Lezcano 1994). Although in prcscntstudy acetic acid content was not determined, the lethal phasealso could be related to greater acetic acid production, whiehunder certain conditions inhibits microbial growth andinduces to the cellular death of the yeast (Ludovico el al.2001). Accumulation of ammonia nitrogen also couldinfluenee yeast death by diminishing aetivity of thedehydrogenase L-glutamate enzyme whieh is neeessary toincorporate nitrogen ammonia into the cell protoplasrn ofthe microorganisms (Metges and Loh 2003) and through thatway make unieellular protein. ResuJts for IVDMD andlVNDFD (Table 1) both inereased significantly (P<0.0005and P<0.004 respectively) at 48 and 72 h of ineubation. DMand OM increased (P<O.OOI and P<0.05 rcspeetively) up to24 h, and then remained constant. This increase could bedue LO the loss of water by evaporation. CP content showedno differenees with the ehange in fermentation time.However, TP increased in the first 24 h (P<0.03) and couldbe related lo the growth of microorganisms, which is inagrcerncru witb Pandey el al. (200 l) stating that TP eould bean indirect way to measure microbial growth in SSFprocesses. NDF eomposition was not affeeted by time offermentation, but ADF decreased (P<O.OOO 1) as thefermentation time passed, probably due to the degradationofthe fibrous fraetion by eellulolytic microorganisms: Valiñoel al. (2002) utilized sugar cane as a substrate in SSF andreponed reduction in tbe level of cellulose and NDF.

In conclusion, the SSF of applc bagasse enhanccd itsnutritive value through Ole growth of mieroorganisms thatincreased true protein and in vítro digestibiliry of dry matterand fiber.

ACKNOWLEDGEMENTS

The authors wish to acknowledge the Fondos Mixtos

Q

(Gobierno del Estado de Chihuahua- CONACYT) for thefinancial assistanec.

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