Research Article | Open Access

Kitti Wirunpan1* , Sakesan Chinwang2, Nareerat Chaikong1 and Charida Pukahuta1

1Department of Biological Science, Faculty of Science, Ubon Ratchathani University, Ubon Ratchathani 34190, Thailand.
2Department of Agriculture, Faculty of Agriculture, Ubon Ratchathani Rajabhat University. Ubon Ratchathani 34000, Thailand.
J Pure Appl Microbiol, 2019, 13 (1): 117-125 | Article Number: 5363

http://dx.doi.org/10.22207/JPAM.13.1.12 | © The Author(s). 2019 

Received: 10/11/2018| Accepted: 22/10/2018 |Published: 20/02/2019
Abstract

This research studied the increasing of nutritional content in cassava starch fermented with P. ostreatus (Jacq.) P.Kumm. and L. squarrosulus Mont. The cassava starch waste was supplemented with maize grain, paddy rice, rice bran, broken rice, and soybean meal at 0, 10, 20, and 30%. Solid state fermentation was carried out for 28 days. The results showed that highest reduced sugar at 86.09 g/L was found in cassava starch waste 70% + rice broken 30% fermented by L. squarrosulus Mont. The highest protein content at 127.92 g/L was found  in cassava starch waste 90% + soybean meal 10% fermented with cassava starch waste 70% + rice broken 30% for 14 days. It is concluded from this experiment that P. ostreatus (Jacq.) P.Kumm. and L. squarrosulus Mont. can increase nutritional content in several feed mixtures.

Keywords

Cassava Starch Wastes, Pleurotus ostreatus (Jacq.) P.Kumm., nutritional content,Lentinus squarrosulus Mont.

Introduction

It is estimated that the world cassava production in the year 2016 can be as high as 288 million tons. This is produced mainly in the continents of Africa, Asia and South America. The production in Thailand is estimated to be 3.2 million tons1 Thailand uses cassava for cassava flour (54%), cassava chip (46%) and ethanol production (2%). By product of the cassava flour production is 10 – 15% cassava starch wastes, and this was used for animal feed but with some limitation due to high fiber and low protein contents2. Several methods were used to increase nutritional contents of this by-product. Some yeast and fungi species were used to increase the nutritional contents by fermentation3,4.  Mushroom classified in the white rot fungi group possess ability to digest lignin and able to increase protein as found in Jonathan et al.5. In these work, P. pulmonarius was used to cultivate with rice straw and sorghum stem. Protein content in rice straw was raised from 4.50% to 9.36% and 5.31% to 8.62% in the sorghum stem5, Bentil et al. also found that cocoa pulp protein content increased from 21% to 26% by using P. ostreatus for 6 weeks6. Darwish et al. also experiment by fermenting corn stem with P. ostreatus for 28 days and found that protein content was raised from 3.6% to 8.15%7. A work by Nasehi et al. using P. florida showed that protein content in rice straw was raised from 3.02% to 7.06% and that in wheat straw was raised from 3.71% to 7.38%8. This study was objected to determine the effect of a solid state fermentation involving P. ostreatus (Jacq.) P. Kumm. and L. squarrosulus Mont. on soluble protein and reducing sugars in cassava starch waste.

Materials and Methods

Experimental Design
The experiment was laid out based on Completely Randomized Design (CRD) with 16 treatments and each treatment contain 3 replicates. This is as follow:

treatment 1 : controls (cassava starch waste 100  %)

treatment 2 : cassava starch waste 90 % + corn 10 %

treatment 3 : cassava starch waste 80 % + corn 20 %

treatment 4 : cassava starch waste 70 % + corn 30 %

treatment 5 : cassava starch waste 90 % + paddy 10 %

treatment 6 : cassava starch waste 80 % + paddy 20 %

treatment 7 : cassava starch waste 70 % + paddy 30 %

treatment 8 : cassava starch waste 90 % + rice bran 10 %

treatment 9 : cassava starch waste 80 % + rice bran 20 %

treatment 10 : cassava starch waste 70 % + rice bran 30 %

treatment 11 : cassava starch waste 90 % + rice broken 10 %

treatment 12 : cassava starch waste 80 % + rice broken 20 %

treatment 13 : cassava starch waste 70 % + rice broken 30 %

treatment 14 : cassava starch waste 90 % + soybean meal 10 %

treatment 15 : cassava starch waste 80 % + soybean meal 20 %

treatment 16 : cassava starch waste 70 % + soybean meal 30 %

The experiment is carried out in 15 x 100 mm petri dishes. Material from treatment 1 – 16 at 30 g was placed inside the dishes and autoclave at 121 degree Celsius for 60 minutes. The mycelia from P. ostreatus (Jacq.) P. Kumm. and L. squarrosulus Mont. was sub-cultured and stored at 27 and 35 degree Celsius, respectively for 14 and 28 days.

Chemical analysis
Reduced sugar: The amount of reduced sugar present in the samples was determined using DNS methodology. The absorbance at 530 nm was measured using a spectrophotometer 9.

Soluble protein: The soluble protein concentrations were evaluated using a colorimetric method based on the standard curve of bovine serum albumin at 750 nm10.

Carbon content: The carbon content was determined following the rapid wet oxidation method11.

Results and Discussion

Reduced sugar
Reduced sugar content in various substrates was found to be significantly different (P<0.05) after fermentation with P. ostreatus (Jacq.) P.Kumm. for 14 days (Table 1). Treatment 13 (cassava starch waste 70 % + rice broken 30 %) showed the highest reduced sugar reading at 16.99 g/L. The lowest reduced sugar content was found in treatment 15 (cassava starch waste 80 % + soybean meal 20 %).

Reduced sugar contents was also found to be significantly different (P<0.05) after fermentation with P. ostreatus (Jacq.) P.Kumm. for 28 days (Table 2). At this period, treatment 10 (cassava starch waste 70 % + rice bran 30 %) showed the highest reduced sugar content (20.28 g/L). Treatment 16 (cassava starch waste 70 % + soybean meal 30 %) was found to contain the lowest reduced sugar reading (0.69 g/L).

Reduced sugar content in all treatments was also found to be significantly different (P<0.05) after fermentation with L. squarrosulus Mont. for 14 days (Table 1). Treatment 12 (cassava starch waste 70 % + rice broken 30 %) showed the highest reduced sugar reading at 22.87 g/L. The lowest reduced sugar content was found in treatment 15 (cassava starch waste 80 % + soybean meal 20 %).

Reduced sugar contents was also found to be significantly different (P<0.05) after fermentation with P. ostreatus (Jacq.) P.Kumm.  for 28 days (Table 2). At this period, treatment 13 (cassava starch waste 70 % + rice broken 30 %) showed the highest reduced sugar content (86.09 g/L). Treatment 15 (cassava starch waste 80 % + soybean meal 20 %) was found to contain the lowest reduced sugar reading (3.84 g/L).

Table 1 
Effect of P. ostreatus (Jacq.) P.Kumm. and  L. squarrosulus Mont. on reduced sugar content at 14 days (g/L)

control P. ostreatus (Jacq.) P.Kumm. L. squarrosulus Mont.
type of substrate 14 day 14day Increasing 14 day Increasing
g/L g/L g/L g/L g/L
cassava starch waste 100 % 0.75defg 14b 13.26a 7.17bc 6.42cd
cassava starch waste 90 % + corn 10 % 1.25cdef 15.61ab 14.37a 11.29abc 10.04abcd
cassava starch waste 80 % + corn 20 % 1.32cde 14.63b 13.31a 15.65abc 14.34abcd
cassava starch waste 70 % + corn 30 % 1.53bcd 15.82ab 14.29a 13.76abc 12.23abcd
cassava starch waste 90 % + paddy 10 % 0.69defg 13.65b 12.97a 8.65bc 7.97bcd
cassava starch waste 80 % + paddy 20 % 0.39fg 14.91b 14.52a 16.89abc 16.50abcd
cassava starch waste 70 % + paddy 30 % 0.21g 16.93ab 16.72a 20.89ab 20.67abc
cassava starch waste 90 % + rice bran 10 % 3.19a 16.48ab 13.28a 14.66abc 11.48abcd
cassava starch waste 80 % + rice bran 20 % 3.93a 19.24a 15.31a 17.75abc 13.83abcd
cassava starch waste 70 % + rice bran 30 % 3.79a 8.36c 4.57b 17.26abc 13.47abcd
cassava starch waste 90 % + rice brokens 10 % 0.56efg 14.75b 14.19a 22.82ab 22.27abc
cassava starch waste 80 % + rice brokens 20 % 0.36fg 15.53ab 15.17a 26.20a 25.84a
cassava starch waste 70 % + rice brokens 30 % 0.35fg 17.34ab 16.99a 23.21ab 22.87ab
cassava starch waste 90 % + soybean meal 10 % 1.59bcd 6.01cd 4.43b 11.82abc 10.23abcd
cassava starch waste 80 % + soybean meal 20 % 2.27b 2.35d 0.075b 2.885c 0.61d
cassava starch waste 70 % + soybean meal 30 % 2.12bc 2.66d 0.53b 3.785c 1.67d

a, b, c, d, e, f, g  = Means in the same column with different superscripts are significantly different (P=0.05)

Table 2 
Effect of P. ostreatus (Jacq.) P.Kumm. and  L. squarrosulus Mont. on reduced sugar content at  28 day (g/L)

control P. ostreatus P.Kumm. L. squarrosulus Mont.
type of substrate 28 day 28day Increasing 28 day Increasing
g/L g/L g/L g/L g/L
cassava starch waste 100 % 0.66b 8.61e 7.95f 8.82hi 8.16ghi
cassava starch waste 90 % + corn 10 % 1.46b 12.03de 10.58ef 31.6e 30.14d
cassava starch waste 80 % + corn 20 % 1.69b 16.89bc 15.2bcd 52.09c 50.41c
cassava starch waste 70 % + corn 30 % 1.76b 13.26cd 11.5def 62.76b 61b
cassava starch waste 90 % + paddy 10 % 0.39b 13.9cd 13.52bcde 21.1fg 20.71def
cassava starch waste 80 % + paddy 20 % 0.44b 11.86de 11.42def 42.86d 42.42c
cassava starch waste 70 % + paddy 30 % 0.26b 13.82cd 13.57bcde 47.2cd 46.95c
cassava starch waste 90 % + rice bran 10 % 1.36b 18.67b 17.32ab 16.52gh 15.17efgh
cassava starch waste 80 % + rice bran 20 % 1.79b 18.82b 17.03abc 18.18g 16.39efg
cassava starch waste 70 % + rice bran 30 % 5.17a 25.44a 20.28a 28.19ef 23.02de
cassava starch waste 90 % + rice brokens 10 % 0.67b 15.97bcd 15.3bcd 63.22b 62.56b
cassava starch waste 80 % + rice brokens 20 % 0.33b 16.08bcd 15.76bc 80.41a 80.09a
cassava starch waste 70 % + rice brokens 30 % 0.32b 13.51cd 13.19cde 86.41a 86.09a
cassava starch waste 90 % + soybean meal 10 % 1.14b 2.15f 1.01g 14.47gh 13.33fghi
cassava starch waste 80 % + soybean meal 20 % 1.3b 2.72f 1.42g 5.13i 3.84i
cassava starch waste 70 % + soybean meal 30 % 1.84b 2.53f 0.69g 8.57hi 6.73hi

a, b, c, d, e, f, g, h, i  = Means in the same column with different superscripts are significantly different (P=0.05)

Reduced sugar content in every treatment increased as the fermentation time increased. Hossain et al. also found reduced sugar content increasing after using P. sajor-caju co-cultivated with bean shell, rice straw, wheat straw, and sugarcane bagasse12. Adamafio et al. also found that cellulose was double after fermenting corn cob with P. ostreatus. Solid state fermentation with P. ostreatus decrease accumulated fiber by enzymes activities13. It is indicated that mushroom contains several enzymes that involves lignocellulose metabolism14. A similar finding was also found by Darwish et al. which used P. ostreatus and Saccharomyces cerevisiae with corn stem7. Bentil et al. also found a similar result when experimented with cocoa pulp.

Carbon content
Carbon content in every treatment showed significantly different (P<0.05) after fermentation for 14 days (Table 3). It was found that cassava starch waste 70 % + corn 30 % showed the lowest reduction in carbon content (5.63%). It was also found that a substrate mixture of cassava starch waste 70 % + rice broken 20 % showed the lowest reduction in carbon content (0.11%).  At day 28 after fermentation, the results were also significantly different (P<0.05) (Table 4). The mixture of cassava starch waste 70 % + soybean meal 30 % found the highest reduction in carbon content (6.21%) and the mixture of cassava starch waste 90 % + rice broken 10 % resulted in the lowest reduction in carbon content (0.14%).

Table 3 
Effect of P. ostreatus P.Kumm. and  L. squarrosulus Mont. on carbon percentage  content at  14 day (g/L)

control P. ostreatus P.Kumm. L. squarrosulus Mont.
type of substrate 14 day 14day reduction 14 day reduction
% % % % %
cassava starch waste 90 % + corn 10 % 47.74ab 42.68 abc 5.06ab 47.11ab 0.63a
cassava starch waste 80 % + corn 20 % 47.62 abc 43.27 abc 4.36 abc 47.42a 0.20 a
cassava starch waste 70 % + corn 30 % 48.33ab 42.70 abc 5.63 a 46.34ab 2.00 a
cassava starch waste 90 % + paddy 10 % 47.06abc 46.48 a 0.58bc 45.99 ab 1.06 a
cassava starch waste 80 % + paddy 20 % 45.4bcd 44.84 abc 0.57bc 43.45 abcd 1.95 a
cassava starch waste 70 % + paddy 30 % 46.42abc 43.92 abc 2.50abc 43.91 abcd 2.51 a
cassava starch waste 90 % + rice bran 10 % 48.25ab 45.34 abc 2.91abc 44.44 abc 3.81 a
cassava starch waste 80 % + rice bran 20 % 46.62abc 46.11 ab 0.51bc 42.83 abcd 3.78 a
cassava starch waste 70 % + rice bran 30 % 48.50a 46.11 ab 2.39abc 44.62 ab 3.88 a
cassava starch waste 90 % + rice broken 10 % 47.63abc 46.32 a 1.31abc 46.96 ab 0.67 a
cassava starch waste 80 % + rice broken 20 % 46.25abcd 46.15 ab 0.11c 45.73 ab 0.53 a
cassava starch waste 70 % + rice broken 30 % 48.4a 45.58 ab 2.84abc 44.64 ab 3.77 a
cassava starch waste 90 % + soybean meal 10 % 44.65cd 44.18 abc 0.48bc 42 bcd 2.65 a
cassava starch waste 80 % + soybean meal 20 % 43.33de 41.19 bc 2.15abc 39.36 cd 3.98 a
cassava starch waste 70 % + soybean meal 30 % 41.03e 40.29 c 0.75bc 38.82 d 2.21 a

a, b, c, d  = Means in the same column with different superscripts are significantly different (P=0.05)

Table 4 
Effect of P. ostreatus (Jacq.) P.Kumm. and  L. squarrosulus Mont. on carbon percentage  content at  28 day (g/L)

control P. ostreatus P.Kumm. L. squarrosulus Mont.
type of substrate 24 day 28day reduction 28 day reduction
% % % % %
cassava starch waste 90 % + corn 10 % 49.02a 45.38bc 3.64ab 47.82a 1.20bc
cassava starch waste 80 % + corn 20 % 48.83ab 47.36ab 1.47bc 46.68ab 2.15abc
cassava starch waste 70 % + corn 30 % 49.29a 47.68a 1.61bc 45.98abc 3.32abc
cassava starch waste 90 % + paddy 10 % 46.04cde 45.67abc 0.37c 45.2abcd 0.84c
cassava starch waste 80 % + paddy 20 % 45.39de 44.49c 0.90c 43.84cd 1.55bc
cassava starch waste 70 % + paddy 30 % 44.81ef 44.04c 0.77c 40.47f 4.35ab
cassava starch waste 90 % + rice bran 10 % 47.99abcd 46.76ab 1.23bc 45.10bcd 2.89abc
cassava starch waste 80 % + rice bran 20 % 48.02abcd 45.87abc 2.15bc 43.52cd 4.50ab
cassava starch waste 70 % + rice bran 30 % 48.17abc 45.95abc 2.22bc 43.19de 4.98a
cassava starch waste 90 % + rice broken 10 % 47.62abcd 47.47ab 0.15c 46.68ab 0.94c
cassava starch waste 80 % + rice broken 20 % 46.3bcde 45.42bc 0.88c 46.07abc 0.23c
cassava starch waste 70 % + rice broken 30 % 48.16abc 47.23ab 0.93c 43.65cd 4.51ab
cassava starch waste 90 % + soybean meal 10 % 42.26bcd 40.88d 1.38bc 40.74ef 1.51bc
cassava starch waste 80 % + soybean meal 20 % 41.99g 36.52e 5.47a 39.21f 2.78abc
cassava starch waste 70 % + soybean meal 30 % 40.80g 34.60e 6.21a 39.37f 1.43bc

a, b, c, d, e, f, g  = Means in the same column with different superscripts are significantly different (P=0.05)

Protein contents
Protein content percentage in all treatments showed significantly different (P<0.05) after fermentation for 14 days (Table 5). It was found that protein content in cassava starch waste 70 % + soybean meal 30 % showed the highest protein content (69.92 mg/g). The mixture of cassava starch waste 70 % + rice bran 30 % showed the lowest protein content (3.93 mg/g). It was also showed significantly different (P<0.05) after fermentation for 28 days (Table 6). The highest protein content (51.43) were found in cassava starch waste 70 % + soybean meal 30 % and the lowest protein content were found in cassava starch waste 80 % + paddy 20 %  (8.31 mg/g).

The experiment with L. squarrosulus Mont. for 14 days also showed significantly different (P<0.05) after fermentation for 14 days (Table 5). The highest protein content (127.92 mg/g) was found in cassava starch waste 90 % + soybean meal 10 %. The protein content in cassava starch waste 90 % + paddy 10 % showed the lowest percentage (3.40 mg/g). At day 28 after fermentation the result also showed significantly different (P<0.05) (Table 6). Cassava starch waste 80 % + soybean meal 20 % gave the highest protein content (100.52 mg/g). The lowest protein content (16.65 mg/g) was found in cassava starch 100 %.

The result in this experiment clearly showed that both species of white rot fungi can increase protein content and other nutrients in cassava starch waste. An experiment by Akinifemi et al15 using P. ostreatus and P. pulmonarius co-cultivate with sorghum for 21 days showed that protein content increased from 2.54 to 4.54 and 4.59 respectively, the cocoa pulp nutrient increased when this was fermented with P. ostreatus for 6 weeks6,16. Bentil et al. study the influence of white rot fungi (P. ostreatus) on chemical compositions in agro-industrial residues and found that protein content sawdust and bagasse increased from 0.91% to 2.49% and 0.39% and 2.99%, respectively16. Shrivastava et al. using P. ostreatus on sorghum straw and found protein content increased from 3.37% to 5.08%17. It was also found that carbon to nitrogen ratio decreased from 77.6 to 40.45 when compared to control without fermentation7. Experiment on corn stem using P. ostreatus by fermentation for 28 days and found that total protein was increased from 3.6% to 8.15%. Protein was increased from mycelium and released enzymes12.

Table 5 
Effect of P. ostreatus (Jacq.) P.Kumm. and  L. squarrosulus Mont. on protein content at  14 day (g/L)

control P. ostreatus P.Kumm. L. squarrosulus Mont.
type of substrate 14 day 14day Increasing 14 day Increasing
g/L g/L g/L g/L g/L
cassava starch waste 100 % 3.75d 9.51f 5.91cd 11.14d 7.39d
cassava starch waste 90 % + corn 10 % 4.63d 19.28de 10.88cd 20.03d 15.41d
cassava starch waste 80 % + corn 20 % 6.88d 21.21cde 11.23cd 22.16d 15.28d
cassava starch waste 70 % + corn 30 % 5.98d 21.83cde 11.51cd 27.23d 21.25d
cassava starch waste 90 % + paddy 10 % 7.48d 14.29ef 7.83cd 10.88d 3.40d
cassava starch waste 80 % + paddy 20 % 5.40d 15.36ef 7.69cd 17.91d 12.51d
cassava starch waste 70 % + paddy 30 % 4.98d 13.54ef 5.68cd 21.82d 16.84d
cassava starch waste 90 % + rice bran 10 % 13.13cd 26.28cd 9.48cd 32.76d 19.64d
cassava starch waste 80 % + rice bran 20 % 21.69c 28.59c 7.25cd 98.96c 77.27d
cassava starch waste 70 % + rice bran 30 % 19.85c 28.54c 3.94d 114.17c 94.31bc
cassava starch waste 90 % + rice broken 10 % 6.21d 14.93ef 9.47cd 21.70d 15.49d
cassava starch waste 80 % + rice broken 20 % 3.96d 19.22de 13.22c 23.35d 19.39d
cassava starch waste 70 % + rice broken 30 % 3.92d 19.30de 12.80cd 22.93d 19.02d
cassava starch waste 90 % + soybean meal 10 % 42.50ab 83.13b 51.05b 170.42a 127.92a
cassava starch waste 80 % + soybean meal 20 % 37.08b 108.03a 61.36a 146.04b 108.96ab
cassava starch waste 70 % + soybean meal 30 % 53.13a 116.88a 62.92a 153.33ab 100.21b

a, b, c, d, e, f  = Means in the same column with different superscripts are significantly different (P=0.05)

Table 6 
Effect of P. ostreatus (Jacq.) P.Kumm. and  L. squarrosulus Mont. on protein  content at  28 day (g/L)

control P. ostreatus P.Kumm. L. squarrosulus Mont.
type of substrate 28 day 28day Increasing 28 day Increasing
g/L g/L g/L g/L g/L
cassava starch waste 100 % 3.04h 13.08d 8.55d 19.69g 16.65g
cassava starch waste 90 % + corn 10 % 6.54ef 20.11d 12.00cd 45.59ef 39.04efg
cassava starch waste 80 % + corn 20 % 7.21e 19.49d 11.24cd 68.66de 61.45cde
cassava starch waste 70 % + corn 30 % 7.31e 23.11d 14.13bcd 103.21ab 95.90ab
cassava starch waste 90 % + paddy 10 % 4.44fgh 17.75d 9.90d 23.18fg 18.74g
cassava starch waste 80 % + paddy 20 % 5.46efg 16.56d 8.89d 27.49fg 22.03g
cassava starch waste 70 % + paddy 30 % 4.69fgh 18.03d 11.57cd 36.79fg 32.10fg
cassava starch waste 90 % + rice bran 10 % 12.88d 26.02d 13.52cd 62.87de 49.99def
cassava starch waste 80 % + rice bran 20 % 15.50c 51.34d 32.15abc 66.05de 50.55def
cassava starch waste 70 % + rice bran 30 % 18.52b 60.46bc 35.69ab 95.53abc 77.01abc
cassava starch waste 90 % + rice broken 10 % 5.35efgh 20.74d 16.55bcd 44.18efg 38.82efd
cassava starch waste 80 % + rice broken 20 % 3.98gh 23.24d 17.57bcd 77.49cd 73.51bcd
cassava starch waste 70 % + rice broken 30 % 3.06h 23.50d 16.38bcd 79.01bcd 75.94abc
cassava starch waste 90 % + soybean meal 10 % 18.00b 54.38c 29.17bcd 82.09bcd 64.09cd
cassava starch waste 80 % + soybean meal 20 % 19.23b 81.41ab 11.20cd 119.75a 100.52a
cassava starch waste 70 % + soybean meal 30 % 23.88a 98.10a 51.43a 116.31a 92.43ab

a, b, c, d, e, f, g, h  = Means in the same column with different superscripts are significantly different (P=0.05)

CONCLUSION

We conclude that Pleurotus ostreatus (Jacq.) P.Kumm. and Lentinus squarrosulus Mont. can increase protein content, and reduced sugar in the substrates with significantly different (P<0.05). Carbon percentage was decreased after fermented with both species. The substrates were able to be used in livestock feed.

Acknowledgments

The authors thank Assistant Professor Dr. Sureerat Butprom for her kind technical advice and valuable comments on the manuscript.

Conflict of Interest

The authors declare that there is no conflicts of interest.

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