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Nutrient quality, digestibility and amino acid of cassava leaves silages in different additives

Azhar Amir, Bagus Priyo Purwanto, Nahrowi, Afton Atabany, Salundik and Ahmad Yani

Center of Competitiveness Development, Ministry of Village, Development of Disadvantaged Regions and Transmigration, Jakarta 12740, Indonesia
Bogor Agricultural University 16680, Indonesia
azhar.amir@kemendesa.go.id

Abstract

This study was conducted in Bogor, West Java, Indonesia, to evaluate the nutritional content, anti-nutritional, digestibility, and amino acid profile of the Malang 4 cassava leaf silage with bitter variety. A completely randomized design was in the experiment with five treatments and four replications. The treatments were: CLS (cassava leaf silage without additive), CLSM (CLS + 5% molasses), CLSC (CLS + 5% cassava meal), CLSHF (CLS + 5% fermented liquid of Hi-fer), and CLSKF (CLS + 5% kfir). Color, odor, pH, moisture, nutrient, LAB population, anti-nutrient, digestibility, in vitro fermentability, and amino acid were measured. Our results showed that there were significant differences in their effects on pH, moisture content, proximate analysis, digestibility, and in vitro fermentation (P<0.05). The quality of silage with the addition of 5% molasses is better than other treatments in terms of physical properties and nutritional content. Addition of 5% kfir to silage improved digestibility and in vitro fermentation of cassava leaves. Each treatment reduced levels of HCN and tannins but did not show significant differences between treatments (P>0.05). The amino acid content of cassava leaves such as methionine, cysteine, and lysine could potentially be digested and absorbed in the intestines of ruminants. Therefore, the inclusion of molasses (a source of carbohydrates) and kfir (probiotics) to ensilage can maintain the nutritional value of the bitter varieties of cassava leaves.

Keywords: bitter, cassava leaf, fermentation, HCN, tannin


Introduction

Cassava (Manihot esculenta Crantz) is one of the staple majors in Indonesia. The total production of cassava in 2018 was over 19.3 million tons with a harvested area of 792 thousand ha (Statistic Indonesia 2018). In general, cassava roots are used as an ingredients food source of carbohydrates (64.8%), the starch industry (20.1%), the animal feed industry (2.6%), other non-food industries (7.9%), scattered (2.7%) and around 1.9% are exported (Center of Data and Information, Ministry of Agriculture 2016). By-products from the starch industry such as cassava chip, cassava pulp, cassava peels, and cassava leaf meal are also used for livestock feeding. The by-product is a mixture of feed ingredients in concentrate. Cassava leaf flour is used as a source of protein for poultry and others as a source of energy for ruminants.

Cassava roots are rich in energy about 75 to 80 % of soluble carbohydrate but low protein about 2 to 3% crude protein (CP) of dry matter (DM) (Wanapat and Kang 2015). Cassava leaves, on the other hand, are high in crude protein content 25.7% (Chhay Ty et al 2011), ranging from 21.5 to 30.3% based on the variety of cassava (Jamil and Bujang 2016). Preston (2002) states that cassava can produce a high yield of protein up to 4 tonnes/ha/year. This value is good as a complementary for the low quality of tropical grass. Thus, the leaves are high appropriated for utilization for ruminants feed.

Cassava leaves area abundant in the harvest season. If it isn't use immediately, it will cause decay. Besides, the leaves contain anti-nutritional factors such as tannins and hydrogen cyanide (HCN).The Presence of tannins over 5% on the dry matter can interfere digestibility of nutrients and feed efficiency (Naumann et al 2017). At high concentrations, HCN is poison and can be lethal to the ruminant. The minimum deadly dose of HCN about 2 mg/kg body weight for sheep and cattle, when obtained in the form of glycoside (Radostits et al 2007). While a study by Chhay Ty et al (2011) has shown intake of HCN in pig diets with no apparent toxicity. However, the anti-nutritional substances must be reduced to avoid disruption of ruminant production. Ensilage is an effective way of decreasing the tannin and HCN concentration in cassava leaves (Sudarman et al 2016a).

One of the successes of making silage is with the addition of additives, especially rich additives carbohydrate. Many additives can be used in making silage. In general, additives can be divided into two, namely: additives that act as stimulating the growth of lactic acid bacteria (LAB) (such as bacteria culture, LAB direct, glucose, sucrose, molasses, potatoes) and inhibiting microorganisms undesirable (such as sulfur acid, acetic acid, etc.) (McDonald et al 1991).

Carbohydrate sources that can be used as an additive to silage should therefore be readily achieved by smallholder farmers. Prebiotics and probiotics can also be used as additives to create acid conditions in the ensilage process. A fermented liquids additive of of Hi-fer (prebiotic) and kfir (natural probiotic) are too easy to find. This research aimed to study the effect of various additives on nutrient, anti-nutrient, digestibility and amino acid of cassava leaves silage.


Materials and methods

The experiment was carried out in the Dairy Nutrition Laboratory of the Faculty of Animal Science, Bogor Agricultural University, West Java, Indonesia, from May to June 2017 in the dry season. The cassava variety in this study was Malang 4 type with a bitter taste and had a concentration of tannins of 5.5% of DM and HCN of 121.6 mg/kg (fresh foliage).

The leaves used to arrive from cassava which has been harvested at the age of 8 to 9 months by farmers in Ciampea Village, Bogor, West Java. Molasses and cassava meal are obtained from animal feed stores, fermented liquid of Hi-fer from the Center for Tropical Animal Studies, and kfir from the laboratory of milk analysis, Bogor Agricultural University. The fermented liquid of Hi-fer is a slightly -colored liquid containing formic acid (HCOOH), organic salts, microbial growth promoters, and antioxidants. According to Suryahadi (2014) that this liquid can accelerate the fermentation process and increase feed palatability. Kfir was inoculated into milk at a ratio of 5 %, incubated at 30oC for a day within the dark, so aseptically filtered through a plastic sieve and cold-stored for continuous culture incubation. The filter product is used as a treatment additive. Kfir contains lactic acid bacteria, Acetobacter, and yeasts which have a pH of 3.8 to 3.9, 2.3% fat, and 12.5% ​​DM.

Silage preparation and experimental design

Fresh leaves are allowed to wilt for a day at ambient temperature until the moisture content reached around 60% of DM. Subsequently, cassava leaves were chopped into 3-5 cm length and 5 kg was mixed thoroughly with 5% of the different additive. Its mixed materials were put into 10 kg of transparent plastic bags and compressed by a vacuum pump to exclude air. The plastic bags were tied and put into a sack to protect silage from damage and stored to allow fermentation for 21 days. After 21 days of fermentation, the plastic bags were opened and the quality of silage was evaluated.

This study used a completely randomized design (CRD) using five treatments with four replicates. The treatments were: CLS = cassava leaves silage without additive, CLSM = CLS + 5% molasses, CLSC = CLS + 5% cassava meal, CLSHF = CLS + 5% fermented liquid of Hi-fer, and CLSKF = CLS + 5% kfir. The variables measured were color, odor, pH, moisture, nutrient, LAB population, anti-nutrient, digestibility, and amino acid.

Variable measurement

The color and odor of silage were estimated through organoleptic observations by 15 respondents, pH values were carried out using the Naumann and Bassler method (1997). The nutritional content of silage was measured through proximate analysis (AOAC 1990). The population of LAB was known by the total plate count (TPC) method of Fardiaz (1992). Tannin levels were measured by the spectrophotometric method while HCN levels were measured by the spectrophotometer method (AOAC 2005). In vitro digestibility, with rumen fluid of dairy cattle, were analyzed using the method of Tilley and Terry (1996). Ammonia concentrations (NH3) were analyzed through diffusion micro techniques (Conway 1962). Volatile fatty acid concentrations (VFA) were analyzed using the steam distillation method (General Laboratory Procedures 1966). Amino acid concentrations were analyzed using the method of High-Performance Liquid Chromatography (HPLC) (AOAC 2005).

Statistical Analysis

Data of color, odor, the population of LAB, and amino acid were analyzed using descriptive statistical analysis. The data for nutrient, tannin, HCN, in vitro digestibility, NH3, and VFA were analyzed using General Lineal Model (GLM) in the IBM SPSS program version 21. Data were expressed an means and standard errors of means (SEM). A significant difference (P>0.05) calculated using Duncan new multiple range test (DMRT).


Results and discussion

Silage quality

All treatments didn't show any decay during the ensiling. Parameters of color, odor, pH, and moisture are shown in Table 1. Based on the color test, the silages in this study were included in a good category. Odor test noted that single treatments with an additive of molasses having a sour smell, while the other groups had a mild sour smell. Macaulay (2004) that good quality silage is shown in light green to yellowish or brownish green and sour smell without rancidity.

Table 1. Physical characteristics of cassava leave silage fermented with different additives

Treatments

Color

Odor

pH

Moisture (%DM)

CLS

Brownish green

Mild sour

4.29bc

73.20a

CLSM

Brownish green

Sour

4.00a

71.12a

CLSC

Brownish green

Mild sour

4.26bc

72.70a

CLSHF

Yellowish green

Mild sour

4.35c

75.55b

CLSKF

Brownish green

Mild sour

4.33bc

73.02a

SEM

0.03

0.55

p

0.000

0.013

Mean values in the same column without common superscripts (a,b,c) differ at p<0.05

The effect of treatments on cassava leaves significantly decreased the pH and moisture content of the silage (P<0.05). The results in the present study have a higher value than that reported by (Sudarman et al 2016b), which has a better pH value and water content after 28 days of fermentation. an additive of molasses showed the best results. However, other treatments were not bad with a pH value <4.5 and a moisture content below 76%. The pH and moisture content of silage above this threshold can cause spoilage and reduce feed intake (Macaulay 2004).

Proximate composition

The proximate analysis of cassava leaves silage with different additive is shown in Table 2. The inclusion of additives in cassava leaves silage was significantly different in dry matter (DM), crude protein (CP), crude fat (CF), and total digestible nutrient (TDN) while the crude fiber was not significantly different. The CLSM treatment showed the best results in DM content.

Table 2. Proximate composition of cassava leave silage fermented with different additives (% DM)

Treatments

DM

DM Basis

CP

EE

CF

TDN

CLS

26.80b

25.92b

5.9b

16.8

66.32bc

CLSM

28.97b

24.06b

3.9a

17.0

63.37ab

CLSC

27.30b

21.80a

4.6a

18.6

62.27a

CLSHF

24.45a

25.72b

6.1bc

20.3

64.14abc

CLSKF

26.98b

25.27b

6.8c

16.8

67.10c

SEM

0.55

0.45

0.26

0.6

0.58

p

0.000

0.004

0.000

0.263

0.022

DM: Dry matter; CP: Crude protein; EE: Extract ether; CF: Crude fiber; TDN: Total digestible nutrient Mean values in the same column without common superscripts(a,b,c) differ at p<0.05

The principle of making silage is to preserve and reduce the loss of nutrients from a forage to be used in the future (Bolsen and Sapienza 1993). The DM with an additive of molasses increased 4.7% from 27.5% of fresh cassava laves. The DM losses occurred in other treatments of 0.7%, 1.9%, 2.5%, 11% (CLSC, CLSKF, CLS, CLSHF, respectively). Loss of dry matter in silage products is caused by the process degradation of Water Soluble carbohydrates (WSC) or easily digested sugars into final products simple like acetic acid, lactic, and butyric acid. The Molasses has a high WSC content of 72.1% (Sudarman et al 2016b), thus preventing to decrease in the DM of cassava leaves. All the silages could be sorted into acceptable quality silages. A Decrease in dry matter obtained in this research is still in limitation normal for a fermented product. According to Lendrawati et al (2012) stated that percentage of dry matter loss on well-managed silage 8% as well McDonald et al (1991) numbers in the range 7-20%.

Anti-nutritional and LAB population

Table 3 shows the content of cyanide, tannin, and the number of LAB in all treatments analyzed. There were no significant differences in the levels of HCN and tannins. The Ensiling process reduced both HCN and tannin contents of cassava leaf, from 121.6 to below 29 mg/kg fresh basis and from 5.5 to below 1.6 % DM, respectively.

Cyanide poisoning is associated with the amount of feed intake and the animals' physiological condition. Intake HCN concentration over 220 mg/kg on fresh forage is dangerous. Forage containing less than 100 mg/kg HCN fresh materials is usually safe to pasture. Forages with an HCN content of more than 500 mg/kg DM should be considered potentially toxic (Cope 2014).

Table 3. Anti-nutrient and LAB of CLS with different additive

Treatments

HCN (mg/kg
fresh basis)

Tannin
(% DM)

LAB
(cfu/g)

CLS

28.14

1.34

3.9x108

CLSM

27.58

1.42

7.9x106

CLSC

27.78

1.39

2.3x108

CLSHF

25.88

1.36

1.6x109

CLSKF

25.95

1.54

5.6x109

SEM

0.36

0.03

p

0.238

0.514

Ruminants are more susceptible to cyanide poisoning than monogastric animals. This is due to the rumen pH 6 to 7 which is alkaline, high water content, and microflora enzymes in the rumen hydrolyze the cyanide glycosides to HCN. This is consistent with Chhay Ty et al (2011) that there was no association between HCN intake and the performance of pig production. The value of low pH in the pig abdomen may de-activate the enzymes that affect the release of the HCN.

Cassava leaves contain condensed tannins which are commonly found in tropical plants. The concentration of tannins obtained in this study was in the range of 1.34 to 1.54%. This level is lower than the report by Phoung et al (2015) in the range of 2 to 2.6% for sweet and bitter types after 24h fermentation. At moderate levels, condensed tannins are known to have positive effects on the nutritional value of the feed by forming insoluble complexes with dietary protein, resulting in the "escape" of rumen fermentation protein.

The fermentation process causes a decrease in the number of bacterial colonies of lactic acid. This is related to the nature of the lactic acid bacteria and its pH produced on the ensilage. The bacterial population in this study exceeds the minimum limit of 105 cfu/g (McDonald et al 1991). According to Lendrawati et al (2012) that the minimum value is achieved at the age of 6 weeks of silage with a pH of 3.8 to 3.9.

In-vitro digestibility, ammonia, and volatile fatty acid

The results showed that the inclusion of additives into cassava foliage silage significantly (P<0.05) affected in-vitro digestibility dry matter (IVDMD), in vitro organic matter digestibility (OMD), ammonia content (NH3), and volatile fatty concentration. The CLSKF treatment with the addition of kfir showed the best results in IVDMD and IVOMD. Kfir is a natural probiotic that contains colonies of LAB and yeast. According to El-Tawab (2016) state that probiotics improve the rumen microbial ecosystem, nutrient digestibility, and feed conversion rate. The digestibility of cassava leaf silage in this study was lower than that reported by Sudarman et al (2016b) with a range of IVDMD of 51 to 65% and IVOMD in the range of 49 to 62%. This is due to differences in the variety and harvest time of cassava root, and rumen fluids used.

The CLSKF treatment has also shown the highest levels of NH3 and VFA (22.87 mM and 154.20 mM, respectively). As a result of rising proteolytic activity, the higher protein content of feed would produce in higher NH3 concentration. NH3 is one of the protein productions in the rumen which is used as the main nitrogen source for the reproduction of rumen microbes. The VFA level produced in this study was still in the normal range for rumen microbial growth in the range 80-160 mM (McDonald et al 1991). Besides, they stated that the output of VFA was also influenced by the carbohydrate sources used.

Table 4. In-vitro digestibility, volatile fatty acid, and ammonia of cassava leaves silage

Treatments

IVDMD (%)

IVOMD (%)

NH3 (mM)

VFA (mM)

CLS

38.24a

34.80a

15.15a

138.50bc

CLSM

41.05b

37.39b

16.35a

150.23bc

CLSC

43.44c

42.07c

17.27a

128.89ab

CLSHF

39.60ab

37.24b

16.79a

111.27a

CLSKF

45.04c

43.74c

22.87b

154.20c

SEM

0.62

0.82

0.76

4.60

P

0.000

0.000

0.002

0.006

IVDMD: In-vitro dry matter digestibility; IVOMD: In-vitro organic matter digestibility; VFA: Volatile fatty acid; NH3: Ammonia
Mean values in the same column without common superscripts (a,b,c) differ at p<0.05

Amino acid concentration

The amino acid profile of cassava leaves silage is presented in Table 5. In general, the amino acid content was higher after fermentation 35d, especially in asparagine, glutamate, lysine, and isoleucine. Our study showed that the amino acid content of cassava leaves silage relatively lower than the amino acids in whole cassava hay, except for similar content of methionine and cysteine. This amino acid content is also relatively lower than the silage of cassava leaves at 3 months of cutting from the study of Nguyen et al (2012). However, the elements of methionine and cysteine; were higher. Further, Methionine + cysteine and lysine are the first limiting amino acids in functional diets formulated for pigs from dried or ensiled cassava leaves. According to Vzquez-Aňn et al (2006) Methionine is strongly necessary for the growth rate and the maintenance of all animals. Methionine acts as a precursor to cysteine and other sulfur bonds.

Table 5. Amino acid profile of Malang 4 cassava leaves on silage treatment with different additives (g/100g)

Amino acid

CLS##

CLSM#

CLSC#

CLSHF#

CLSKF##

Asparagine

1.78

0.78

0.78

0.79

1.58

Glutamine

2.68

1.95

2.13

1.71

2.31

Serine

0.35

0.34

0.36

0.44

0.29

Glycine

0.26

0.75

0.49

0.63

0.27

Histidine

0.47

0.32

0.34

0.27

0.54

Arginine

0.51

0.44

0.37

0.38

0.58

Threonine

0.75

1.03

0.24

0.35

0.69

Alanine

0.47

0.73

0.13

0.21

0.41

Proline

1.34

0.51

0.45

0.96

1.15

Tyrosine

0.71

0.37

0.50

0.24

0.69

Valine

0.65

0.80

0.53

0.74

0.51

Methionine

0.51

0.50

0.26

0.51

0.48

Cysteine

0.46

0.42

0.11

0.49

0.39

Isoleucine

0.76

0.61

0.63

0.58

0.82

Leucine

0.81

1.01

1.11

1.05

0.91

Phenylalanine

0.62

0.36

0.50

0.28

0.61

Lysine

1.12

0.51

0.93

1.00

0.92

# after fermentation 28d; ## after fermentation 35d


Conclusion


Acknowledgment

Financial support from the Indonesian Directorate General of Higher Education, Ministry of Research, Technology and Higher Education through the Leading Research program 2017.


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