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Fermenting dried maize cobs with the fungus Pleurotus eryngii increased the content of crude protein and in vitro gas production

Nguyen Thi Huyen1,2, Nguyen Thi Tuyet Le1 and Bui Quang Tuan1

1 Department of Animal Nutrition and Feed Technology, Faculty of Animal Science, Vietnam National University of Agriculture, Trau Quy, Hanoi, Vietnam
nthuyencnts@gmail.com
2 Animal Nutrition Group, Wageningen University, The Netherlands

Abstract

The objective of this experiment was to determine the nutrients value and total gas production of dried maize cobs (DCC) after fermented with the fungus Pleurotus eryngii. In experiment 1, the dried maize cobs was treated with P. eryngii at the concentrations of 2.5% of wet weight of substrate for 2 and 4 weeks. Each experimental treatment was done in 3 replicates. Results showed that fungal treatment increased the CP content and reduced NDF, ADF and ADL of the DCC. The content of NDF and ADL was lowest, while the content of CP was highest when incubating the DCC with P. eryngii for 4 weeks. In experiment 2, the DCC and fungal treated DCC were incubated for 72h in the buffer rumen fluid mixture. Results showed that total gas production after 72h incubation was highest when the DCC was treated with P. eryngii for 4 weeks. Based on the results mentioned above, it could be concluded that Pleurotus eryngii can be used to improve nutrients value of dried maize cobs in Vietnam.

Keywords: Pleurotus eryngii, dried maize cobs, nutrients value, wood-rot fungi


Introduction

In 2020, five million tonnes of maize were produced in Vietnam. Besides, the primary source, a large amount of by-products such as maize stover, maize cobs are produced. However, maize cobs have a low nutrients value. Consequently, most of the maize cobs has been leaved on the field or burnt directly. This is a cause for environmental pollution and waste the renewable source of roughage for ruminants. In addition, maize cobs contains acid detergent lignin (ADL), a large group of recalcitrant aromatic polymers bonded with hemicellulose by ether and ester linkages, forms a matrix that tightly surrounds the cellulose. This complex lignocellulosic structure prevents the fermentation process by rumen microbes and hydrolyzation by cellulose (Van Kuijk et al. 2015). Therefore, it is necessary to break down the bonds between ADL and hemicellulose by treatments to release the cellulose from this matrix and enhance the feed utilization rate of maize cobs.

Physical and chemical treatment of maize stover and maize cobs to increase the intake and digestibility by ruminants studied for decades ago. However, Tuyen et al (2012) stated that physical and chemical treatments can be expensive, harmful to users or unfriendly to the environment. Biological methods using white-rot fungi may be a more viable alternative to improve the nutritional value of rice straw. This method is environmentally friendly and potentially economical (Tuyen et al 2012).

Using white-rot fungi to treat maize stover, maize cobs, rice straw and wheat straw has been studied by Zuo et al (2019), Akinfemi (2010), Jafari et al (2007), Akinfemi & Ogunwole (2012), Shrivastava et al (2012) and El-Bordeny et al (2015). The results of Huyen et al (2019) indicated that the nutritional value of rice straw was improved when incubated for 4 weeks with Pleurotus eryngii. The following experiment was conducted to confirm Pleurotus eryngii also can be used to improve nutrients value of maize cobs in Vietnam.


Materials and methods

Fungi treatments of 5-mm dried maize cobs for 2 weeks and 4 weeks

The experiment was conducted at Department of Animal Nutrition and Feed Technology, Vietnam National University of Agriculture, Hanoi, Vietnam from October to January 2018. The dried maize cobs (DCC) was collected in Mai Chau, Son La province, Vietnam. The DCC was ground in a cross-beater mill to pass through a 5mm sieve. After that, the DCC was soaked in water for 24 h. The soaked DCC was then removed and drained of water for 24 h. One kilograms (fresh basis) soaked DCC was packed in polyethylene bags (40 cm length and 20 cm diameter and 2.54 mm thickness), that was immediately tied up with a little cotton on the top of bag by nylon rope. The bags were autoclaved for 1 h at 121 C. The autoclaved DCC bags were cooled at 20 C and then were inoculated with spawn at 2.5% of rice straw (fresh weight basis). The DCC was fermented with Pleurotus eryngii (P. eryngii; strain MES 03757) according to the procedure developed by Tuyen et al (2012). All the bags were transferred to the fermentation room, which was maintained at 30 C and the relative humidity of the room was maintained at 75 % for 2 weeks and 4 weeks. Then all bags were removed from the fermentation room and the fungal treated DCC was oven-dried at 65 C for 3 days. The DCC and fungal treated DCC were ground in a cross-beater mill to pass through a 1mm sieve, then was stored at 4C before analysis of DM, ash, N, NDF, ADF and ADL and using for gas production experiment.

The fungus was sourced from the Wageningen UR Plant Breeding Center, the Netherlands.

Table 1. Chemical composition of dried maize cobs (raw) and dried maize cobs (autoclaved)

Items

Dried Maize cobs
(Raw)

Dried Maize Cobs (Autoclaved)

DM

946.3 1.4

954.2 2.9

Chemical composition (g/kg DM)

OM

899.1 1.7

902.6 2.5

CP

61.2 1.4

56.3 1.3

NDF

740.8 2.0

754.6 5.5

ADF

463.8 1.8

485.9 6.0

ADL

89.3 1.7

102.7 2.1

In vitro gas production experiment

In vitro gas production was conducted according to the method described by Menke et 5 al (1979). In summary, a mixture of rumen fluid was collected before feeding time in the morning from three different rumen fistulated lactating Holstein-Friesian dairy cows. These cows were fed a grass and maize silage mixture and concentrate according to their requirements 2 times per day. The rumen fluid was filtered through four layers of cheesecloth into pre-warmed thermo flasks. A strict anaerobic condition was maintained during rumen fluid collection. Buffer solution was made as described in the method of Cone et al (1996). The rumen fluid was mixed with a buffer solution in a 1:2 (v/v) ratio under a continuous flux of CO2. Approximately 500 mg of the oven-dried DCC (control) and fungal treated DCC substrates were weighted triplicates into 100-mL calibrated glass syringes. The grass syringes were pre-warmed at 39C before adding with 60 mL of the buffer rumen fluid mixture then they were incubated in a water bath at 39C. Three blank glass syringes only contained 60 mL of the buffer rumen fluid mixture. The gas production was manually recorded at 0, 1, 2, 4, 6, 8, 12, 24, 36, 48, 60 and 72h. The gas production was calculated by subtracting the mean of gas production from three blank syringes and expressed on an OM basis.

Total cumulative gas production (GP) curves were fitted with a monophasic Michaelis-Menten equation of Groot et al (1996), using the non-linear least squares regression procedure in SAS (SAS, 2010).

Where OMCV = Gas production (mL/g of incubated OM), A = the asymptotic gas production (mL/g of incubated OM), B = the switching characteristics of the curve, C = time at which half of the asymptotic gas production is reached (half-time, T, h) and t= the time (h).

Measurements sampling and analytical procedures

The DCC (raw and autoclaved) and fungal treated DCC samples were analysed for DM, ash and nitrogen according to AOAC (2005) methods. Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) were determined according to Van Soest et al (1991).

Statistical analysis

The different of chemical composition, gas production at 72h and fermentation kinetics of the DCC and fungal treated DCC were analysed by ANOVA using the MIXED procedure of SAS (SAS, 2010). The model was:

Y = μ + Ti + εij (2)

where Y = the dependent variable, μ = the overall mean, Tj = the effect of treatment (i=1 to 3) and εij=the residual error term. The results are presented as the least squares means and standard error of the means. Differences among main effects were analysed using Tukey-Kramer’s multiple comparison procedure in the LSMEANS statement of SAS (SAS 2010) with effects considered significant at p≤0.05 and a trend at 0.05<p

Table 2. Chemical composition of dried maize cobs (autoclaved) and fungal-treated dried maize cobs after 2 and 4 weeks

Items

Control

2 weeks

4 weeks

SEM

p

DM

954.2a

891.9b

834.9c

1.97

<.0001

Chemical composition, g/kg DM

OM

902.6a

830.7b

767.5c

2.19

<.0001

CP

56.3a

72.2b

82.5c

0.68

<.0001

NDF

754.6a

654.0b

615.3c

2.47

<.0001

ADF

485.9a

449.7b

427.6c

2.39

<.0001

ADL

102.7a

88.8b

70.3c

0.85

<.0001

abc Row means with different superscripts differ at p<0.05



Table 3. The loss of elements from dried maize cobs after incubation with fungi for 4 weeks

Items

Dried Maize
Cobs (g)

Fungal-treated Dried
Maize Cobs (g)

Loss/gain
(%)

DM

100.0 0.0

87.5 0.65

-12.5 0.65

OM

94.6 0.39

80.4 0.46

-15.0 0.42

CP

5.9 0.15

8.7 0.08

+46.8 5.12

NDF

79.1 0.80

64.5 0.16

-18.5 0.95

ADF

50.9 0.75

44.8 0.36

-12.0 0.64

ADL

10.8 0.25

7.4 0.11

-31.5 0.78



Table 4. Fermentation kinetics of dried maize cobs and fungal-treated dried maize cobs after 2 and 4 weeks

Items

Control

2 weeks

4 weeks

SEM

p

Total GP (72h, ml/g incubated OM)

141.2a

160.6b

177.9c

0.91

<.0001

A, (ml/g incubated OM)

226.9a

266.4b

276.6c

2.31

<.0001

B

0.84a

0.88b

0.84a

0.004

0.0005

C (half-time, h)

37.0a

40.8b

32.9c

0.79

0.0013

abc Row means with different superscripts differ at p<0.05; A = the asymptotic gas production (mL/g of incubated OM), B = the switching characteristics of the curve, C = time at which half of the asymptotic gas production is reached (half-time, h)



Figure 1. Gas production of dried maize cobs and fungal-treated dried maize cobs during 72h incubation


Results and discussion

Fungal treatment increased the CP content of the DCC (Table 2 and Table 3). Similar results were reported by Zuo et al (2019) who reported that CP content of maize stover was increased from 61.3 to 74.5 g/kg DM when it was treated with Irpex lacteus for 28 days. Similar improvements in CP content of fungal treated maize cobs were also found by Akinfemi (2010). The CP content increased from 3.89% (control) to 10.11% forPleurotus ostreatus treated maize cob and 7.46% for Pleurotus pulmonarius treated maize cob (Akinfemi, 2010). Huyen et al (2019) found that the CP content improved by Pleurotus eryngii treated rice straw for 28 days. The CP increase could be due to the increased fungal biomass and also affected by the loss of substrate during the fermentation Huyen et al (2019).

The concentrations of ADF, NDF and ADL were reduced by fungal treatment. Similar results were reported by Zuo et al (2019), Akinfemi (2010), Vorlaphim et al. (2018) and Huyen et al (2019). The fungi require substrates such as cellulose, hemicellulose or other carbon sources for their growth, the end products being fungal protein and carbon dioxide, the latter accounting for the overall 12.5% loss of substrate DM during the fermentation (Table 3). Huyen et al (2019) reported 14.8 % DM loss when rice straw was fermented with Pleurotus fungi. On the other hand, the decreasing of NDF, AFD and ADL content in the fungal treated samples could be due to the ability of the fungi to secrete hydrolyzing and oxidizing enzymes, which could aid the decomposition of recalcitrant compounds in maize cobs into utilizable compounds. This observation confirms that maize cobs, which are very low in nutrients, could be successfully become ruminant feeds after treated with fungi.

Total gas production after 72h incubation was highest when the DCC was treated with Pleurotus fungi for 28 days (Table 4 and Figure 1). These findings are supported by in vitro studies of Zuo et al (2019) and Tuyen et al (2013). The higher gas production in the fungal treated samples could be related to the higher CP content and lower content of NDF, ADF and ADL compared to the control samples (Osuga et al. 2006). The lower content of fibre in the fungal treated samples can facilitate the colonization of the feed by the rumen microbial population, which in turn might increase the fermentation rate, therefore improving digestibility. As the fermentation process is partially regulated by the fibre content of the feeds, the fungal treated maize cobs fermented faster than the untreated.


Conclusions


References

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