EFFECT OF SOLID SUBSTRATE FERMENTATION ON THE NUTRITIONAL QUALITY OF AGRO-INDUSTRIAL RESIDUES

Guadalupe Ortiz-Tovar, Javier López-Miranda, María Andrea Cerrillo-Soto,

Arturo Juárez-Reyes, Ernesto Favela-Torres and Oscar Soto-Cruz

María Guadalupe Ortiz-Tovar. M.Sc. in Biochemical Engineering, Instituto Teconológico de Durango (ITD), Mexico. Manager, Agrana Fruit de Mexico.

Javier López-Miranda. M.Sc. in Food Engineering, ITD, Mexico. Professor, ITD, Mexico.

María Andrea Cerrillo-Soto. Ph.D. in Animal Nutrition, Iowa State University, USA. Professor, Universidad Juárez del Estado de Durango (UJED), Mexico.

Arturo Saúl Juárez-Reyes. Ph.D., Université Pierre et Marie Curie, Paris VI, France. Professor, UJED, Mexico.

Ernesto Favela-Torres. Ph.D., Université de Provence, France. Professor, Universidad Autónoma Metropolitana (UAM), Unidad Iztapalapa, Mexico.

Nicolás Oscar Soto-Cruz. Doctor in Biotechnology, UAM, Mexico. Professor, ITD, Mexico. Address: Blvd. Felipe Pescador 1830 Ote., Col. Nueva Vizcaya, 34080, Durango, Dgo., Mexico. e-mail: soto@itdposgrado-bioquimica.com.mx

This study was performed to evaluate the chemical composition and in vitro gas production of five agro-industrial residues, with and without treatment, using solid substrate fermentation. Oat straw, bean straw, corn stubble, apple pomace and agave bagasse were treated using two filamentous fungi, Trichoderma harzianum and Phanaerochaete chrysosporium. Chemical analyses (crude protein, neutral detergent fiber, acid detergent fiber, lignin, cellulose and hemicellulose) were carried out. Calibrated glass syringes were used for in vitro gas production determinations and volatile fatty acid (VFA) profile was obtained by gas chromatography. In vitro data were fitted using the equations p=a+b(1-e^-ct) by Ørskov and McDonald and G=A/(1+[B/t]) by France et al (2000). Increases in CP content (P<0.05) were registered by treating oat straw (20%), bean straw (83%) and corn stubble (16%) with P. chrysosporium. Contents of NDF were reduced by 9.5% in oat straw by treatments (P<0.05). Regarding gas production parameters, the b value in agave bagasse (57.4%) and gas production rate constant c (7.5%) were higher (P<0.05) in the substrate treated with P. chrysosporium related to other treatments. Similarly, higher values for the A parameter were registered in maize straw (+22%) and agave bagasse (+13%) treated with P. chrysosporium. In spite of these isolated differences, the analysis performed showed that the chemical composition, mainly structural carbohydrates, were not modified under the conditions of this study.

EFECTO DE LA FERMENTACIÓN EN MEDIO SÓLIDO SOBRE LA CALIDAD NUTRICIONAL DE RESIDUOS AGROINDUSTRIALES

RESUMEN

El objetivo de este trabajo fue evaluar la composición química y la producción de gas in vitro de cinco residuos agro-industriales, antes y después de aplicar un proceso de fermentación en medio sólido. Paja de avena, paja de haba, rastrojo de maíz, bagaso de manzana y bagaso de agave fueron tratados usando dos hongos filamentosos, Trichoderma harzianum y Phanaerochaete chrysosporium. Los análisis químicos realizados fueron: determinación de proteína cruda, fibra detergente neutro, fibra detergente ácido, lignina, celulosa y hemicelulosa. Para la determinación de la producción de gas in vitro se utilizaron jeringuas de cristal calibradas, mientras que el perfil de ácidos grasos volátiles (AGV’s) fue obtenido por cromatografía de gases. Los datos de producción de gas in vitro fueron ajustados usando las ecuaciones p=a+b(1-e^-ct) propuesta por Ørskov y McDonald y G=A/(1+[B/t]) propuesta por France et al (2000). Se observaron aumentos en el contenido de proteína cruda (P<0,05) al tratar paja de avena (20%), paja de haba (83%) y rastrojo de maíz (16%) con P. chrysosporium. El contenido de fibra detergente neutro fue reducido un 9,5% en paja de avena por los tratamientos (P<0,05). Con respecto a los parámetros de la producción del gas, los valores de b y c en bagaso de agave fueron mayores (57,4% y 7,5%, respectivamente) en el substrato tratado con P. chrysosporium en relación con otros tratamientos. De manera similar, valores más altos para el parámetro de A fueron observados en rastrojo de maíz (+22%) y bagaso de agave (+13%) tratados con P. chrysosporium. A pesar de estas diferencias aisladas, el análisis realizado demostró que la composición química, principalmente carbohidratos estructurales, no fue modificada bajo las condiciones de este estudio.

RESUMO

O objetivo deste trabalho foi avaliar a composição química e a produção de gás in vitro de cinco resíduos agroindustriais, antes e depois de aplicar um processo de fermentação em meio sólido. Palha de aveia, palha de fava, resíduos de milho, bagaço de maça e bagaço de agave foram tratados usando dois fungos filamentosos, Trichoderma harzianum e Phanaerochaete chrysosporium. As análises químicas realizados foram: determinação de proteína crua, fibra detergente neutro, fibra detergente ácido, lignina, celulosa e hemicelulose. Para a determinação da produção de gás in vitro se utilizaram seringas de cristal calibradas, enquanto que o perfil de ácidos grassos voláteis (AGV’s) foi obtido por cromatografia de gases. Os dados de produção de gás in vitro foram ajustados usando as equações p=a+b(1-e^-ct) proposta por Ørskov e McDonald e G=A/(1+[B/t]) proposta por France et al. Se observaram aumentos no conteúdo de proteína crua (P<0,05) ao tratar palha de aveia (20%), palha de fava (83%) e resíduos de milho (16%) com P. chrysosporium. O conteúdo de fibra detergente neutro foi reduzido um 9,5% em palha de aveia pelos tratamentos (P<0,05). Com respeito aos parâmetros da produção do gás, os valores de b e c em bagaço de agave foram maiores (57,4% e 7,5%, respectivamente) no substrato tratado com P. chrysosporium em relação com outros tratamentos. De maneira similar, valores mais altos para o parâmetro de A foram observados em resíduos de milho (+22%) e bagaço de agave (+13%) tratados com P. chrysosporium. Apesar destas diferenças isoladas, a análise realizada demonstrou que a composição química, principalmente carboidratos estruturais, não foi modificada sob as condições deste estudo.

KEYWORDS / In Vitro Gas Production / Phanaerochaete chrysosporium / Trichoderma harzianum /

More than 60 million tons of oat straw, bean straw, maize straw, apple pomace and agave bagasse are produced in Mexico annually, being the main agro-industrial residues produced in the country (Soto-Cruz, 2003). The main components of agro-industrial residues (cellulose, hemicellulose and lignin) are complex and its biodegradability is low, due to their resistance to degradation by ruminal microorganisms (Jarrige et al., 1995). Nevertheless, biotechnology could offer opportunities to modify the chemical structure of these substrates and to improve their digestion. Some efforts have been made to apply biotechnological processes to the improvement of the nutritious quality of agro-industrial residues like ruminant feed. Villas-Boas et al. (2003) treated apple pomace using Candida utilis in submerged culture, followed by Pleurotus ostreatus in solid substrate fermentation (SSF). They found that after C. utilis fermentation, protein and mineral content increased 100 and 60%, respectively, accompanied by an 8.2% increase in digestibility, while sequential fermentation with C. utilis and P. ostreatus achieved a high protein level with 500% of crude protein enrichment after 60 days of fermentation, as well as a considerable increase in the mineral content. Bauer et al. (2003) evaluated the effect of enzymatic treatments on several substrates rich in carbohydrates, showing that the fermentabilities of the enzyme-treated and untreated substrates were different. Thus, the utilization of SSF, which requires sophisticated technology and is of low cost (Ghiydal et al., 1992) offers the potential of improving the nutritional value of agro-industrial residues. SSF has been defined as the growth of the micro-organisms on (moist) solid material in absence or near-absence of free water (Pandey, 2003). Under these conditions, the favored microorganisms are filamentous fungi, due to its capacity to grow in media with low water activity (Cannel and Moo-Young, 1980).

On the other hand, in vivo evaluation of feed quality is expensive and laborious. Thus, simple and economical techniques are desirable (Stern et al., 1997), like in vitro gas production (Williams, 2000), which is used to determine the fermentation kinetics of cereal straws, cereal grains (Opatpatanakit et al., 1994) and other feed (Blümmel y Becker, 1997; Getachew et al., 2004). Moreover, the gas produced during in vitro incubation can be related to production of short chain VFA (Getachew et al., 2002), which are the main energy source for ruminants. This technique has not been used to study the quality of roughages treated by solid substrate fermentation.

The purpose of the present study was to evaluate the effect of SSF on the nutritional quality of agro-industrial residues, through chemical analysis, gas production and VFA production in vitro.

Agricultural residues comprising oat straw, bean straw, maize straw, apple pomace and agave bagasse were used as substrates. All the substrates were milled through a 1mm screen and stored in sealed plastic containers for further analyses.

Two strains of filamentous fungi (Trichoderma harzianum and Phanaerochaete chrysosporium), obtained from the collection of the Universidad Autónoma Metropolitana-Iztapalapa were used. The strains were stored at 4°C in plates with potato dextrose agar (PDA).

Fermentation was carried out in duplicate using 250ml Erlenmeyer flasks and 4g of the residue. A solution containing (per liter) 0.3g urea, 1.4g (NH₄)₂SO_4, 2.0g KH₂PO₄, 0.4g CaCl₂·2H₂O, 0.3g MgSO₄·7H₂O, 0.75g casein peptone, 0.25g yeast extract, 5mg FeSO₄·7H₂O, 1.6mg MnSO₄·4H₂O, 1.4mg ZnSO₄·7H₂O, and 20mg CoCl₂·6H₂O was added to obtain a humidity of 38%. Flasks were sterilized at 120°C during 15min. As soon as flasks reached room temperature, they were inoculated (final humidity of 75%) to provide the following concentrations (spores/g DM) in three treatments: 1) 2×10⁷ spores/g of T. harzianum, 2) 2×10⁷ spores/g of P. chrysosporium, and 3) 1×10⁷ spores/g of each of the two fungi. After inoculation, all the flasks were incubated for 7 days at 30ºC.

Crude protein was determined by the Kjeldahl method (AOAC, 1995). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and lignin were determined according to Van Soest et al. (1991). Hemicellulose was calculated as (NDF-ADF) and cellulose as (ADF-lignin).

Samples (200mg DM) were placed into 100ml calibrated glass syringes. Buffered and mineral solutions (Menke and Steingass, 1988) were combined in a 2:1 proportion with rumen fluid collected from three rumen fistulated sheep fed alfalfa hay and a commercial concentrate (75:25). Thirty ml of buffered rumen fluid was dispensed to each syringe. The syringes were shaken by hand at 1h intervals in the initial 3h of incubation. Reading of gas volumes were recorded at 0, 3, 6, 9, 12, 24, 48, 72 and 96h of incubation.

Using another set of syringes, after 24h of incubations, ~10ml of syringe contents were sampled followed by centrifuging at 1000´g during 20min. Of the supernatant, 5ml were pipetted into a 10ml plastic tube containing 1ml of 25% metaphosphoric acid solution and centrifuged at 1000´g for subsequent VFA determination by gas chromatography (Getachew et al., 2004).

Analysis of in vitro gas production data

All the analyses were made by triplicate. The models used were fitted using the tool Solver^® of Microsoft Excel 2000^®. The results obtained for the models’ parameters, VFA production and chemical composition were analyzed by ANOVA test of one factor using P<0.05. This analysis was made in a spreadsheet of Microsoft Excel 2000^®.

The chemical composition of the substrates without treatment and treated by solid substrate fermentation (SSF) are shown in Table I. Significant increases (P<0.05) were registered in the content of crude protein (CP) for the oat straw (20%), maize straw (16%) and bean straw (83%), when P. chrysosporium was used in the SSF. This can be explained by the fungal growth, which transforms sugars and non-protein nitrogen into CP, as observed previously (Villas-Boas et al., 2003). Increases (P<0.05) in the neutral detergent fiber (NDF) content in apple pomace (11%) and the increase in hemicellulose content in oat straw (26%) and apple pomace (134%), in relation to non-treated substrate were also registered. Table II shows the values obtained when the parameters of the models were fitted by the Ørskov and France models. In 85% of the cases, the adjustment made with the model of Ørskov and McDonald (1979) was better than the adjustment made with the France (France et al, 2000) model. Nevertheless, high values of the r² coefficient were obtained when using both models. The two models are similar, since A and b have exactly the same meaning (the maximum theoretical volume of gas produced from the substrate, which is reached asymptotically), while B and c are related to the rate of gas production.

The parameter B represents the time necessary to reach half of the asymptotic gas production, and it is equivalent to the constant of Michaelis-Menten (K_M). Thus, a low value of B indicates a high affinity of the ruminal microflora by the substrate employed. The parameter c has been interpreted as the constant rate of gas production, which indicates the availability of nutrients of a substrate in the rumen (Blümmel and Becker, 1997); a large value of c indicates high availability of substrate. This can be seen as another form of affinity between rumen microflora and substrate.

The larger values of A (64.08 to 96.90ml per 500mg DM) and b (54.3 to 71.38ml per 500mg DM) were registered usually in substrates without treatment, with the exception of agave bagasse treated with P. chrysosporium, where an increase in the value of A (10%) was observed (P<0.05). Regarding the c parameter, higher values (P<0.05) were registered in substrates such as oat straw, bean straw and agave bagasse without treatment; whereas similar values were obtained in corn stubble and apple pomace with or without treatment (P>0.05). Higher B values were registered in all treated substrates (P<0.05) compared to those without treatment, with the exception of corn stubble, which showed no difference (P>0.05). Moreover, the results indicate a close negative relationship between the c parameter in the Ørskov’s model and the B value in the France’s model (r= -0.91). Overall, the data might indicate that the residues without treatment have better nutritional characteristics than the treated residues. Blümmel and Becker (1997) reported higher b values (33.6-74ml) in a study using degradation characteristics of 54 straws; however, their rates of gas production (c) were smaller than the values found in the present study. Rodrigues et al. (2002) indicated that there were no differences (P<0.05) when analyzing wheat straw without treatment and adding urea, using Orskov´s and France’s models.

Villas-Boas et al. (2003) reported increases in the digestibility and nutritious quality of apple pomace treated sequentially with Candida utilis and Pleurotus ostreatus. The results obtained in the present study agree with those obtained by Bauer et al. (2003), who evaluated the effect of enzymatic treatments on several rich carbohydrate substrates using in vitro gas production and France´s model. They found that enzymatic treatments applied to sugar cane pulp and wheat straw diminished A and increased b (P<0.05).

Ruminants utilize VFA as an energy source, allowing the production of meat and/or milk. Table III shows the molar proportions of VFA. The results indicate significant (P<0.05) changes in the oat straw when SSF treatments were applied. The proportion of acetic acid was greater, while the proportion of propionic acid decreased, when the oat straw was treated with P. chrysosporium. Van Houtert (1993) indicated that relatively large amounts of propionic acid are reported when easily digestible substrates are supplied to rumen microbes. This scenario is probably contrary to what occurred in the present study, where poor cell contents in the studied forages led to a proportionally lower propionate production. SSF treatments did not modify the molar proportions of VFA in the cases of agave bagasse, apple pomace, maize stubble and bean straw. These results agree with those of Bauer et al. (2003), who determined the effects of diverse treatments (measuring in vitro gas production) to improve the quality of foods for ruminants. They applied enzymatic treatments to sugar cane pulp and bean straw, and found that the enzymatic treatment applied to the sugar cane pulp did not produce differences (P>0.05), but in the case of the bean straw receiving enzymatic treatment a decrease in total VFA occurred. Getachew et al. (2004) analyzed several ruminant foods using in vitro gas production, indicating no difference (P<0.05) between the proportions of VFA for substrates without treatment and substrates treated by silage.

The data presented indicates that the chemical composition, mainly the structural carbohydrates, were not modified in the conditions of this study. As a result, neither the in vitro gas production characteristics nor the VFA profile were improved by the treatment with SSF of the evaluated agro-industrial residues. Thus, direct use of agro-industrial residues for ruminant feed, without microbial or enzymatic treatments, is recommended.

Financial support for this work was provided by the project DGO-2002-C01-2041 from the FOMIX-Durango. A scholarship was provided by CONACYT, Mexico to the first author.

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