Production of Microbial Biomass Protein by Arachniotus ruber using Banana Peel and its Biological Evaluation in Broiler Chicks.
Summary: The major issue of the developing countries, i.e. malnutrition, results from intake of diet deficient in proteins. The problem can be resolved by searching for novel and cheap protein sources. Bioconversion of inexpensive fruit waste to an economical product like microbial protein can be useful in the present situation. The aim of the present study was to enhance the crude protein content of banana peels by Arachniotus ruber under solid state fermentation for utilization in broiler feed. Different physicochemical parameters were optimized to increase the yield. Biological evaluation was carried out by replacing 25 and 50% of sunflower meal in broiler feed with the biomass protein. Different parameters e.g.: weight gain, feed efficiency, feed conversion ratio and protein efficiency ratio were recorded. The optimum conditions to achieve highest amount of crude protein (15.8%) on fermentation of banana peels was 1:1.5 substrate water ratio, 2mL inoculum volume, pH 4 after 72 hours of incubation time.
Addition of glucose (0.5%), corn steep liquor (0.25%) and MgSO4 (0.05%) enhanced the yield. Outcome of the study revealed that biomass protein produced, can replace up to 50% of the protein supplied by sunflower meal with the positive effect on weight gain of the chicks.
Keywords: Banana peels, Arachniotus ruber, Microbial protein, Solid state fermentation, Biological evaluation, Broiler chicks.
Malnutrition, the prime issue of the developing countries results from the intake of protein deficient diet. This deficit is expected to rise in upcoming years due to the gap between demand and supplied amount of protein diet. So it is imperative to explore the novel and cheap protein sources to overcome the situation. Since the quality of microbial protein (also called as single cell protein) is considered equal to animal protein, there is growing interest in its production and utilization . Single cell protein refers to dead and dried cells of microorganisms or the proteins extracted from them. It can be produced by cultivating a variety of microorganisms, e.g: algae, fungi and bacteria on the cheap carbon sources. In addition to high protein content, it contains a small fraction of nucleic acids, carbohydrates, fats, minerals and vitamins.
Microbial protein also contains essential amino acids that are lacking in most animal and plant foods. Microbes have the ability to grow rapidly on cheap substrates like agricultural wastes, resulting in cost-effective protein production method instead of utilizing expensive sources such as soybean and fish .
Different fruit processing industries generate tons of waste that have no useful application. It can serve as a cheap carbon source for production of single cell protein. Banana (Musa paradisiaca) fruit peel, an organic waste, contains abundant amount of carbohydrate and other basic nutrients that supports the microbial growth. In tropical climates, such as in Pakistan, the banana trees continue bearing fruit throughout the year. Utilization of this cheap and easily available substrate have advantage of lowering the production cost and also solve the waste disposal and management problems, conserve natural resources and provide feed for livestock purposes . Arachniotus sp, a fungus, used in study to increase the nutritional potential of banana peels has also been reported for the same purpose previously on different agricultural wastes .
The present research was carried out with the aim to increase the crude protein content of banana peels by fermentation with Arachniotus ruber through solid state fermentation and its utilization in poultry feed instead of sunflower meal. This is the first report on consumption of microbial protein, produced by fermentation of banana peels in feed of broiler chicks.
Microorganism and maintenance of culture
The pure and certified culture of Arachniotus ruber, already reported  was acquired from the stock cultures of NIBGE, Faisalabad, Pakistan. The microorganism was cultured and maintained on slants of Potato Dextrose Agar (PDA) at pH 4 and sub cultured at monthly intervals.
Procurement of raw materials
Banana peel samples were obtained from the cafeteria of University of Veterinary and Animal Sciences (UVAS), Lahore. The peels after separation were dried in oven, ground and then sieved through 1-mm mesh screen. Sample was further packed in transparent Zip-lock polythene bags and stored at room temperature for further experimentation .
The peels sample was analyzed for dry matter, crude protein, crude fiber, crude fat, ash and nitrogen free extract .
Preparation of inoculum media
Arachniotus sp was raised on PDA slants for five days. Finally, two loops of spores were suspended in 10 mL of normal saline solution. Then the spores were smashed with the sterile wire loop to break the clumps. Further, the spore count was done with haemocytometer and diluted to obtain the inoculum of 1x106 spores per mL .
Optimization of parameters for biomass protein production
The basal medium for fermentation contained (g/L): CaCl2. 2H2O, 0.05; MgSO4.7H20, 0.05; KH2PO4, 0.1 and corn steep liquor, 0.1. Banana peel (10 g) was used as the substrate and the media was autoclaved at 121AdegC for fifteen minutes. Firstly, the substrate water ratio (1:1, 1:1.5 and 1:2) and incubation time (24, 48, 72, 96 and 120 hours) were optimized after addition of 2 mL inoculum to the flasks at pH 4, 37AdegC . Then volume of inoculum was optimized by using 1, 2, 3, 4 and 5 ml of the inoculum . The effect of addition of carbohydrate was tested by using 0.5% glucose, fructose, sucrose, lactose and starch . Corn steep liquor was utilized as nitrogen source and different concentrations were used to obtain maximum crude protein production (0.1, 0.15, 0.2, 0.25 and 0.3%). Different ionic salts like CaCl2. 2H2O, MgSO4.7H2O and KH2PO4 in varying concentrations (0.025, 0.05, 0.075 and 0.1%). were also checked to get high titer of biomass protein .
Proximate analysis of banana peels and biomass Protein
The proximate analysis of the banana peels was performed before and after fermentation to determine the percentage of crude protein .
Large scale production and amino acid analysis of fermented material
The large scale production of microbial protein was carried out in 5 litre flasks under the pre-optimized conditions. Amino acid analysis of fermented material was performed by amino acid analyser (biochrom Cambridge, UK Bio30+) at Sadiq feeds Private Limited, Rawalpindi, Pakistan.
One hundred broiler chicks were housed in Animal room of the UVAS, Lahore. Day old broiler chicks were fed on starter ration for the period of one week and vaccinated for New Castle disease and Infectious Bursal disease at regular intervals. For biological evaluation, the chicks were selected after one week according to weight uniformity basis and divided randomly into three groups A, B and C based on the rations fed. Each group had three replicates containing ten chicks.
* Group A, control group, was fed on 23 % crude protein diet for five weeks.
* Group B was fed on diet in which 25% of sunflower meal was replaced by biomass protein for five weeks.
* Group C was fed on stock diet by replacing 50% of sunflower meal with biomass protein for five weeks.
At the end of the trial, biological evaluation of the biomass protein was determined in terms of weight gain, feed efficiency, feed conversion ratio and protein efficiency ratio .
The data was statistically analyzed using one way ANOVA technique under CRD (Cohort-CoStat-2003 software version 6.33) while the mean comparison was done with Tukey's Multiple Range test .
Results and Discussion
Proximate analysis of the substrate
The proximate analysis of the banana peel on dry weight basis before fermentation is shown in Table-1.
Table-1: Proximate analysis of banana peels (% Dry weight).
###Crude Protein###4.2 +- 0.5
###Crude Fat###14.56 +- 0.6
###Crude Fibre###11.95 +- 0.32
###Ash###14.58 +- 0.43
NFE (Nitrogen Free Extract represents sugars)###50.37 +- 0.6
Optimization of substrate water ratio and incubation time
The significantly (p<0.05) highest amount of crude protein (7.4%) was achieved at 1:1.5 substrate water ratio in comparison to the control (4.2%), followed by 1:2 (6.6%) and 1:1 (6.1%) after 72 hours of incubation time by fermentation of banana peels (Fig 1). A significant increase in CP content was observed up to 72 hours and then decreased on further incubation. The results indicated the crucial role of moisture content on increasing the CP content of banana peels. Too much moisture content can interfere with the gas exchange resulting in generation of anaerobic conditions, whereas too less can inhibit the fungal growth.
The optimum substrate water ratio depends on a number of factors like water holding capacity, quality and particle size of the substrate. Present results are in line with Shahzad and Rajoka , as higher production of biomass protein (6.09 g/L) was obtained after 72 hours of incubation using broken rice as substrate by Aspergillus terreus. The results differed from Shahzad et al. , as maximum CP (3.12%) was reported at 1:2 substrate water ratio using wheat straw at fourth day of incubation by Arachniotus species. Nadeem et al.  reported maximum yield of biomass (9.8 g/L) after 96 hours of incubation using defatted rice polishing as substrate by Candida utilis.
Effect of volume of inoculum
The above experiment was carried out under pre-optimized conditions with different volumes of inoculum to increase the percentage of crude protein. The significantly highest (p<0.05) crude protein content (7.4%) was obtained by using 2ml of inoculum from spore suspension (Fig. 2). The data indicated increase in CP percentage on addition of inoculum up to 2mL and then decreased further.
The reason may be that increase in number of microorganisms results in the exhaustion of nutrients rapidly. This inhibits the fungal activity resulting in lower yield of protein on fermentation. The results are in line with Oshoma and Ikenebomeh , as they obtained higher production of biomass protein from rice bran using 2% (v/v) inoculum size of Aspergillus niger. The results differed from Irfan et al. , they suggested 10% (v/v) inoculum size to be optimum for production of biomass protein (47.4%) using wheat bran as substrate by Candida utilis. Similarly, inoculum size of 7.5% (v/v) was observed best for the highest production of microbial protein from Candida utilis . Nadeem et al.  reported 5% inoculum size (v/v) to achieve the maximum biomass of Candida utilis (10.2 g/L).
Effect of carbohydrate source
Addition of carbohydrate in the fermentation media had the positive effect on the increase of crude protein content of banana peels. The amount of crude protein achieved in control was 7.4% while addition of carbohydrate source affected in the following order: 0.5% glucose gave the highest yield of crude protein (8.41%) followed by sucrose (8.12%), fructose (7.98%), lactose (7.74%) and starch (7.31%) as indicated in Fig 3. The reason of increase in yield is that carbohydrates are used by the fungus for growth and maintenance. Among all the sugars tested, glucose is most readily sugar consumed by the fungus for its growth and increase in crude protein percentage of banana peels. The results are in line with Munawar et al. , as they analyzed that all the carbon sources except maltose increased the biomass production of Candida utilis and highest dry biomass (2.07g/100ml) was obtained by utilizing soluble starch followed by glucose.
The positive effect of glucose was stated by Adoki  for production of crude protein by utilizing orange plantain and banana processing residues as substrate.
Effect of Nitrogen source
The effect of addition of corn steep liquor to the fermentation media had the positive effect on crude protein content of banana peels. Crude protein (%) increased in parallel to increase in concentration of corn steep liquor up to 0.25% corn steep liquor (12.61%) in comparison to the control (8.41%) and then decreased on further addition of nitrogen source (Fig 4). The reason may be that on further increase in corn steep liquor concentration, the fungal ability to biodegrade banana peels and enhance its growth decreases. The present results are supported by Shahzad et al. (2016), they observed increased CP (3.94%) by addition of 0.15% urea using wheat straw as substrate. Shahzad and Rajoka, (2011) reported corn steep liquor to be the best nitrogen source to achieve high amount of biomass protein (5.09 g/L) on broken rice by using Aspergillus terreus.
Optimization of Ionic Salt concentration
The different ionic salts were tested in varying concentrations to increase the crude protein content of banana peels. The maximum increase in crude protein was obtained by addition of 0.05 % MgSO4.7H2O (15.8%) in comparison to the control (12.61%). Thereafter, it decreased on further increase in the ionic salt concentration. The calcium and potassium ions (0.075% and 0.05%) also had the positive effect on CP content (14.4 and 15%) as indicated in Fig. 5. Shahzad et al.  reported highest amount of CP (3.77%) by addition of 0.05% MgSO4. 7H2O, 0.075% CaCl2 and 0.15% KH2PO4 to wheat bran using Arachniotus species. Jaganmohan et al.  suggested 0.1% MgSO4 to be optimum for Aspergillus terreus biomass production by using Eichornia and banana peel as substrate.
Nadeem et al.  observed best biomass protein production (9.15 g/L) by addition of 0.0025% MgSO4 and 0.025% CaCl2 while Asad et al. , indicated 16.24% biomass protein production by using corn cob substrate on addition of 0.10% MgSO4, 0.05% CaCl2 and 0.3% KH2PO4 using Arachniotus sp.
Amino acid analysis
The amino acid profile of the biomass protein produced on large scale by using banana peel is discussed in Table-2.
The different parameters observed after biological trials are represented in Table-3.
Table-2: Amino acid profile of biomass protein produced on banana peels by Arachniotus ruber.
###Serial number###Amino acid###Banana Peel (%)###Biomass (%) produced on banana peels
Table-3: Biological evaluation of Biomass protein produced by Arachniotus sp against the standard diet.
###Parameters###Ration A###Ration B###Ration C
###Weight gain per chick (g)###1009.05 +- 0.12c###1021.2 +- 0.16 b###1038.8 +- 0.13 a
###Feed consumption (g)###2030.5 +- 0.21 c###2037.7+- 0.16 b###2059.5+- 0.26 a
###Feed efficiency (gain/ feed)###0.497+- 0.09a###0.501+- 0.11a###0.504+- 0.13a
###Feed Conversion Ratio (feed/ gain)###2.01+- 0.06 a###1.99+- 0.09 a###1.98+- 0.07 a
###Protein efficiency ratio (gain/ protein)###3.15+- 0.05 a###3.13+- 0.04 a###3.10+- 0.08 a
The significantly highest weight gain (1038.8 g) was observed in the group of chicks fed on ration C, in which 50% sunflower meal was replaced by biomass protein, then the chicks fed on ration B (1021.2 g) containing 25% replacement of microbial protein with sunflower meal and the lowest in control ration A (1009.05 g). Hence, it is suggested that sunflower can be replaced partially with microbial biomass protein without any deleterious effect on the weight gain of broiler chicks. The results are in accordance with Shahzad and Rajoka ), they utilized biomass protein from Aspergillus terreus in feed of broiler chicks. Joshi et al.  reported similar results by mixing fermented apple pomace with a standard broiler feed in the ratio of 1:1 as compared to the standard feed.
The highest feed consumed by chicks was ration C, while the lowest feed consumed was ration A. The present results are supported by Shehzad and Rajoka, , as they indicated maximum feed consumed was on ration in which soybean meal (60%) was replaced by microbial protein.
Feed efficiency (gain/feed):
Analysis of variance showed non-significant results on comparison of feed efficiency between different rations. Hence, it is concluded that 50% of sunflower meal can be replaced with microbial protein. Similarly, Shehzad and Rajoka  suggested that soybean meal in broiler feed can be substituted successfully up to 60 percent with biomass protein of Aspergillus terreus.
Feed Conversion Ratio:
Analysis of variance showed non-significant difference among the experimental rations. The outcomes of trial are in agreement with Shehzad et al. , who observed non-significant differences in group of chicks nourished on diet in which soybean meal was replaced by biomass protein.
Protein Efficiency Ratio:
The protein efficiency ratio in the present study, according to analysis of variance, differs non-significantly among various experimental diets. The similar outcomes were achieved by the biological evaluation of biomass protein conducted by Shahzad and Rajoka .
The waste banana peels can be utilized as a cheap protein source in broiler feed after processing through fermentation. It contained all the essential amino acids and showed better weight gain in group of broiler chicks fed in comparison to the control diet.
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|Author:||Yasin, Muhammed Shiraz; Saeed, Shagufta; Tayyab, Muhammed; Hashmi, Abu Saeed; Awan, Ali Raza; Firyal|
|Publication:||Journal of the Chemical Society of Pakistan|
|Article Type:||Technical report|
|Date:||Apr 30, 2019|
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