Cassava starch fermentation wastewater: Characterization and preliminary toxicological studies

a, S.R.P. Avancini a,*, G.L. Faccin a, M.A. Vieira a, A.A. Rovaris a, R. Podesta b c a R. Tramonte , N.M.A. de Souza , E.R. Amante
a

ncias Agra ncia e Tecnologia de Alimentos Admar Gonzaga, rias, Departamento de Cie Universidade Federal de Santa Catarina, Centro de Cie polis, SC 88034-001, Brazil 1346, Bairro Itacorubi, Floriano b ncias Biolo gicas, Departamento de Morfologia, Brazil Universidade Federal de Santa Catarina, Centro de Cie c rio, Laborato rio de Ana lises Cl nicas, Brazil Universidade Federal de Santa Catarina, Hospital Universita

Abstract Cassava starch fermentation wastewater is an industrial residue composed mainly of lactic acid bacteria with predominance of the genera Lactobacillus, and organic acids. To evaluate the safety of this residue for possible production of probiotic beverages, acute in mice and sub-chronic (28-day repeated dose) toxicity studies in rats were carried. The administration of a single dose of 5 g/kg/body weight did not produce mortality in mice. Rats treated with water containing 0 (control), 25%, 50%, and 100% of the residue for 28 days, did not present alterations in behaviour or in food and water consumption. There were no treatment-related changes of toxicological signicance in the relative weight of the organs neither in the haematological nor in the biochemical parameters. Histopathologic alterations observed in the small intestine did not seem to be associated with the treatment.

Keywords: Cassava; Fermentation; Wastewater; Toxicity

1. Introduction Cassava (Manihot esculenta Crantz) is largely used in human and animal nutrition, as well as raw material for several industrial products; the most important are the cassava our, the cassava starch and the sour cassava starch. The sour cassava starch production (Fig. 1), begins with the extraction of the cassava starch, which consists of cleaning, peeling, chopping, pressing and straining of the cassava roots. Fibre is separated from the starchy water (starch milk) and the starch is separated from water either by decantation or by centrifugation, according to the industry production capacity; smaller industries employ decantation while bigger industries use the centrifugation process. The cassava starch is then either dehydrated (Vil-

ela and Ferreira, 1987), or submitted to natural fermentation for the production of a sour cassava starch (Demiate et al., 1999). This fermentation occurs empirically, based only on the producers practical knowledge. It is a submerse fermentation, with a supercial water layer of 20 cm and, depending on the region and season, it may take from 30 to 90 days. The fermentative process starts with the sugar production from starch due to amylolytic microorganisms; this step is accelerated by glucose addition (Marcon et al., 2006). From this source of carbon, bacteria and yeasts start the production of acids, aromatic compounds and vitamins among several other substances. Typical microorganisms of the cassava starch fermentation are the lactic acid bacteria with predominance of the genera Lactobacillus (L. plantarum, L. fermentum, L. delbrueckii and Lactobacillus L. manihotvorans), followed by Streptococcus, Enterococcus, Leuconostoc, Pediococcus and Lactococcus (Morlon-Guyot et al., 1998; Lacerda

Cassava roots

Water

Washing

Wastewater

Desintegration Water Separation (First stage)

Recycled liquor

Separation (Second stage)

Pulp wastewater

Starch milk

Water

Screening

Alternative route

Sedimentation

Wastewater

Water

Fermentation

Commercial cassava starch

Sun drying

Wastewater
Sour Cassava Starch
Fig. 1. Flowchart of sour cassava starch production.

et al., 2005; Silveira et al., 2003). Yeasts such as Galactomygua-similar ces geothricum, Issatchenkia scutulata var. ex ndida ethanolica have already been identied. and Ca According to Oyewole (2001), yeast seems to have an important role in the survival and in the activity of lactic acid bacteria during the cassava fermentation process, as they are involved in the cassava starch hydrolysis into simple sugars, which are converted into organic acids by the lactic acid bacteria. The typical organic acids found in the cassava starch fermentation are: lactic, butyric, acetic, and propionic acids. Lactic acid corresponds for around 6080% of such acids (Demiate et al., 1999). The wastewater resulted from the cassava starch fermentation process is rejected. In the concept of end-of-pipe technologies, it is considered as euent with high chemical and biochemical oxygen demand (COD and BOD, respectively). In the concept of Cleaner Production, this residue from cassava starch fermentation presents an interesting

composition that could be used in new products such as probiotic beverages, or in other applications, depending on its composition. Because this water is traditionally considered wastewater, toxicological evaluation is practically inexistent. To evaluate the safety of the cassava starch fermentation water, acute toxicity study was carried out in mice, and sub-chronic (28-day repeated dose) toxicity study was carried out in rats. There is no absolute criterion for selecting a particular animal species (Chan and Hayes, 1994) in toxicological analysis. Ke et al. (2005) used mice in the acute and rats in the chronic toxicity tests, for the evaluation of an antioxidant beverage eective microorganisms-X. Rats, mice, rabbits and guinea pigs are most commonly chosen for acute toxicity studies (Brito, 1994; Chan and Hayes, 1994; OECD, 1995). The use of animal for hazard identication in human risk assessment is far from ideal, but is still widely considered (Barlow et al., 2002).

2. Material and methods

A blend of commercial cassava starch with the same production year from several regions of Santa Catarina State, with the maximal of two months of storage, at environmental temperature, around 25 C, was used in this study.

2.1. Fermentation conditions, physicochemical and microbiological determinations

Cassava starch was submitted to natural fermentation in plastic vast of 564 mm 371 mm 385 mm containing 6.4 kg of starch, 24 L of water and 140 g of glucose. The fermentation was performed for 17 days, until the minimal acidity of 20 mL of N NaOH per 100 mL, detected by titration, was achieved (AOAC, 1998). The water was removed, ltered in cheese whey lter and stored in glass bottle under refrigeration (4 C). Evaluation of mesophilic and lactic bacteria, moulds and yeast in the wastewater was carried out at the end of fermentation, according to APHA methodology (APHA, 2001). The evaluation of acidity and the bacterial count were also carried out 30 days after storage in order to verify the stability during the toxicological assay. Cassava starch fermentation wastewater has shown low concentration of total solids, containing mainly proteins, sugars and lactic acid, as well as low pH (Table 1). The lactic acid bacteria and total mesophilic count was about 106 CFU/mL at the beginning the experiment and also after 30-day under refrigeration. The moulds and yeast count was at 107 in the beginning and at 106 after the 30-day period. For acute oral toxicity study the wastewater was freeze-dried by an E.C. Micromodulyo freeze dryer and kept under refrigeration (4 C) until the analysis. For the repeated dose 28 days study of the wastewater was utilized in the liquid form.

essary to reach dosage. Mice were randomly divided into two groups of six animals. Each group was housed in plastic cages with three animals per cage. The freeze-dried cassava starch fermentation wastewater was suspended in distilled water, and administered orally (1 mL/100 g body weight) to the animals, by gavage, at a dose of 5 g of freeze-dried cassava fermentation wastewater/kg body weight. Control group received distilled water only. Clinical signs of toxicity (alteration in hair, skin and mucous, behaviours, tremors, diarrhoea, convulsions, respiration, cyanosis and others) were observed for the following periods: 5 and 30 min, 1, 2, 4, and 24 h and daily for 14 days. Since food and water was provided ad libitum throughout this period, total food and water intake per cage was measured and the average intake per mouse was determined. The individual weight of the mice was monitored daily. After this period, animals were submitted to euthanasia, in an isolated room, in ether saturation chamber and the liver was removed and weighed (Brito, 1994).

2.4. Sub-chronic oral toxicity or repeated dose 28 days

The doses for the repeated dose study of 28 days were established according to the possible form of consumption of this wastewater, that is, added to another liquid (2550%) or ingested directly as a beverage (100%). Rats were assigned to four groups of 10 animals (ve males and ve females), individually housed in stainless-steel cages. The test groups received the water from cassava starch fermentative process (cassava wastewater) in the drinking water, ad libitum, at three dierent concentrations (25%, 50%, and 100%) and the control group received potable water for 28 days. The drinking water was renewed daily and the consumption evaluated and expressed as mL/kg body weight/day. Body weights were measured twice weekly during all the experimental period. Food intake was registered twice a week. Behaviour and clinical signs were observed throughout the experiment. Hematological and serum biochemical, examinations, as well as necropsy were performed on the 28th day, when blood samples were collected by cardiac puncture under ether anesthesia after 16 h starvation. At necropsy, the major organs: liver, kidneys, heart, spleen and lungs were removed, weighed and compared with the body weight of the rats (relative organ weight) (Brito, 1994; OECD, 1995).

2.2. Animals
This study was approved by the Animal Ethical Committee of The Santa Catarina Federal University. Mice (Mus musculus), female, weighing 26.5 1.0 g were used for the acute oral toxicity because conventional LD50 tests surveys show that females are generally slightly more sensitive (OECD, 1997). Wistar rats of both sexes, weighing 67.64 8.48 g (males) and 65.5 8.15 g (females) were used for the repeated dose study of 28 days. The animals were obtained from the Central Biotery of Santa Catarina Federal University, and maintained for one week in groups of 3 5 animals, in plastic cages of 410 mm 340 mm 180 mm, with stainlesssteel covers on chipped hardwood bedding. Throughout the experiment, the animals were kept in a room under controlled temperature (22 2 C) and a light/dark cycle (12/12 h) with free access to food and water.

2.5. Hematological and serum biochemistry

The following hematological parameters were evaluated in an automated haematological analyser ABX PENTRA 120 (HORIBA ABX Diagnostics) in blood samples collected using EDTA (ethylenediamine tetracetic acid) as anticoagulant: leucocytes (LEU), erythrocytes (ERY), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular haemoglobin concentration (MCHC), platelets (PLT), mean platelet volume (MPV), lymphocytes (LYN), monocytes (MON), neutrophils (NEU), eosinophils (EOS), basophils (BAS). Prothrombin activation time (PAT) analyses were carried out using a Sysmex CA-1500 automated blood coagulation analyser (Sysmex America, Inc.) in blood samples collected in tubs containing sodium citrate as anti-coagulant. Cholesterol (CHOL), low density lipoprotein (LDL), high density lipoprotein (HDL), triglycerides (TGL), creatinine (CREA), alkaline phosphatase (ALP), glucose (GLU), aspartate transaminase (AST), alanine aminotransferase (ALT), total proteins (TP), albumin (ALB), sodium (Na), potassium (K) and chloride (Cl) were determined in blood serum after centrifugation (2500 rpm by 15 min), using a clinical chemistry system DADE Dimension RXL (Dade International Inc., Newark, DE 19714, USA).

2.3. Acute toxicity test

Cassava starch fermentation wastewater is a material with low solid content. For acute toxicity test the sample was freeze-dried. Mice were used in this study due to the small amount of freeze-dried material nec-

Table 1 Chemical composition of cassava starch fermentation wastewater Components Total solids Lipids Protein Ash Carbohydrates Acidity in lactic acid pH Mean DP from triplicate. g/100 mL 0.22 0.04 Non detected 1.35 0.26 0.02 0.001 0.19 0.03 0.19 0.01 3.20 0.17

2.6. Histological analysis

For this preliminary study slices of 2 cm in length, removed from the small intestine in a point located at 5 cm distal from TREITZ angle, were immersed in a sodium chloride aqueous solution at 0.9% and xed in

formaldehyde solution at 10%. The slices were submitted to the process of paran inclusion, sectioned in microtome for histological cuts of 10 lm in thickness, according to the same plane of transversal cut. The laminas were coloured using the haematoxylineosin method. Lamina analysis was carried out on a conventional optical microscope. The parameters used for microscopic evaluation were the presence or not of damaged tissue, such as: loss of epithelial covering (light lesion); inammatory inltrate in lamina propria of mucous (moderate lesion); intestinal villous destruction with presence of inammatory inltrate (intense lesion) (Tramonte et al., 2004).

relative) was veried among the groups. The acute oral LD50 was greater than 5 g/kg of body weight 3.2. Subchronic oral toxicity study No deaths were observed and as for the general conditions of the animals, there were no signicant changes among the control and the test groups. The weight, food and water consumption of the animals that received dierent concentrations of cassava wastewater were not dierent from the control group (Table 2). The mean daily intake of cassava wastewater given as total solids was, respectively, 87.2, 168.9 and 320.2 mg/kg/day in female rats, and 80.6, 168.7 and 318.2 mg/kg/day in male rats (Table 2). A reduction of solid intake was observed with the growth of the rats. Only the relative weight of the lungs from females that received the 50% dose was greater in comparison to the control group. The relative liver weight of the males from the 100% group was smaller than the control group. During blood collection for the analyses, some animals were lost or had their blood lysed. In respect to examination of haematological parameters, only the hemoglobin value of males that received 100% cassava wastewater was signicantly lower in comparison to the control group. No statistically signicant changes were observed in the other parameters examined. For males, CHOL was signicantly elevated in 50% dose, TGL was higher in 25% and 50% dose, GLU was higher in all doses while ALB and Na in 25% dose. However AST was signicantly elevated in 25% and 50% dose and TGL was lower in 50% dose for females. No statistically signicant changes were observed in the other parameters examined. Male animals had light histological lesions on analysed intestinal regions; only one animal from the group that received the highest dose showed intense lesion. However, all the females from the control group as well as from the

2.7. Statistical analysis

All the results are expressed as mean and standard deviation. The weight gain and the liver weight in acute toxicity assay were analysed by the Student t-test. The comparison between sub-acute groups was carried out by one-way analysis of variance (ANOVA) considering signicance level of p < 0.05 with Dunnet post test for comparison with the control. Verication of the model suppositions was carried out by residues analysis and the Bartlet test. When heterogeneity of variances occurred the nonparametric test of KruskalWallis was used, followed by the Mann Whitney test. All the statistical analyses were carried out with the aid of the Software Statistica version 6.0 (2001).

3. Results 3.1. Acute toxicity Throughout the 14 days of assay, the test group did not show any change in behaviour, mortality, or clinical signs of toxicity compared to the control group. Animals that received the testing product (5 g/kg of body weight) showed a smaller weight gain in comparison to the control group. A loss in weight occurred in the test group after ingestion of the product. However, this group immediately regained weight, but only reaching around 59% of the control groups nal weight. The test group mean food consumption was lower than the control group, however the liquid was higher. No signicant dierence in relation to liver weight (absolute or

Table 2 Mean weight gain and food and water consumption and cassava wastewater intake of rats exposed to 0%, 25%, 50% or 100% cassava wastewater in the drinking water for 28 days Dose (%) Female 0 25 50 100 Male 0 25 50 100 Mean weight gain (g) 116.2 8.2 105.0 12.7 106.6 7.0 109.2 14.0 180.0 8.6 200.2 16.1 173.9 11.5 192.9 12.2 Mean Feed consumption (g/kg body weight/day) 169.0 8.1 166.6 9.2 173.2 5.1 165.7 8.5 179.8 8.6 179.6 5.5 173.2 1.9 176.7 6.9 Mean water consumption (mL/kg body weight/day) 143.7 17.2 158.6 21.0 153.6 16.2 145.5 15.7 164.9 33.6 146.5 9.5 153.4 6.9 144.6 6.3 Mean cassava wastewater intake (mg/kg body weight/day)a 0.0 87.2 11.5 168.9 17.8 320.2 34.6 0.0 80.6 5.2 168.7 7.6 318.2 35.8

Values are means SD. n = 5. a g total solids from cassava wastewater/kg body weight calculated as follows: water consumption (mL/kg/d) total solids of cassava wastewater (g/100 mL) in the concentration used.

group of the highest dose showed intense lesions, showing intestinal villous destruction with presence of inammatory inltrate in the mucosa and the animals that received the other doses practically did not show any lesions. 4. Discussion This was a preliminary study realized with the aim to evaluate the use of the cassava starch fermentation wastewater, an industrial residue composed mainly of lactic acid bacteria of the genera Lactobacillus and organic acids, at its natural form, in future studies for new beverage development, without drying or concentration process. Since this residue has never been evaluated, an acute toxicity assay in mice and repeated dose 28-day oral toxicity study in rat was realized in order to estimate the toxicological potential of the material. The results obtained in show that the cassava starch fermentation wastewater is of low acute toxicity in mice (>5000 mg/kg of body weight). Considering that humans are more frequently exposed to lower doses when compared to the dose that causes acute toxicity, and for long periods of time, the repeated dose study or sub-chronic toxicity provides more realistic data on toxicity (Lu, 1996; Barlow et al., 2002). The ingestion of the concentrations of cassava starch fermentation wastewater in this study did not produce any eects on the growth of the animals neither on their consumption of water or food. Alterations in the blood biochemical parameters as cholesterol, triglycerides, glucose, albumin and sodium in males; triglycerides, glucose and aspartate transglutaminase in females, showed not to be dose-dependent, and are within the normal rate for the species (Wolford et al., 1986). The values for creatinine, total proteins, albumin, sodium, potassium and chloride in this assay are below the reference values for these animals (Wolford et al., 1986; Charles River Laboratories, 1998); however, they did not show any other clinical signs related to these low values. Only male animals that ingested the dose of 50% showed a sodium serum level higher than that of the control group. According to Meeks (1989), the eects of one compound on biochemical parameters must be evaluated by comparison with a control group, due to the large variation between experimental animals, making it dicult to utilise reference values. The relative organ weight is fundamental to diagnose whether the organ was exposed to the injury or not. The heart, liver, kidney, spleen and lungs are the primary organs aected by metabolic reaction caused by toxicants. The liver is the major site of foreign compounds metabolism in the body (Dybing et al., 2002). Spleenomegalia and hepatomegalia are indirect indicators of infections and bacterial invasion (Tsai et al., 2004). The change observed in the relative weight of the organs did not show a dependence dose, it can represent a normal variation and not as sign of toxicity since the relative weight of the organs

is not related to any other parameter. The small intestine is the part of the gastrointestinal tract where the majority of digestion takes place and the gut mucous is important in host defence. The ability to disturb this mechanical barrier is an indicator of potential pathogenesis for most pathogens and toxigens. Despite the intense lesions (intestinal villous destruction with presence of inammatory inltrate in the mucosa) detected through histopathologic analysis of the intestine of the female animals, it is not possible to establish that the ingested beverage provoked these lesions since the control group also showed such lesions. Inammatory intestinal diseases are common in laboratory animals, frequently associated with bacterial, virus and parasite and acknowledged ethiology in several cases (McClure et al., 1978). More studies are need to know the causes of this lesions and to evaluate the eect of this residue in the others intestinal regions as the large intestine. Ke et al. (2005) evaluated the toxicity of a antioxidant beverage derived from fermentation of unpolished rice, papaya, and sea weeds with lactic bacteria, yeast and photosynthetic bacteria; they did not nd any signicant changes on body weight, food consumption, behaviour, haematological and biochemical parameters, as well as on heart, liver, spleen and lung histological inspection, in rats fed for 90 days with doses of 150, 100 and 50 times the recommended daily dose. Toxicity study of 28 days in rats treated with 2000 mg of dehydrated microorganisms per kg of body weight did not show adverse eect related to the treatment (Kitano et al., 2004). Toxicity evaluations, for their utilisation as a probiotic, carried out with several pure strains of lactic acid bacteria showed that even in large quantities they do not cause adverse eect when analysed in rats (Huang et al., 2003; Tsai et al., 2004a, 2004b). Results from this preliminary study show that the cassava starch fermentation wastewater is of low toxicity; a study carried out for a longer period of time and with a higher number of animals may be necessary for the safety of utilisation of such wastewater, as well as to evaluate possible probiotic properties. References
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