Fermentation of Selenium-added coconut dregs improve chicken egg production and slow down the deterioration of egg quality during 28 days storage

U Hatta, S Mozin, A Adjis and B Sundu

Animal Husbandry Department, Tadulako University, Palu, Central Sulawesi, Indonesia, 94118
ummianihatta71@gmail.com

Abstract

A study was conducted to determine the influence of selenium-added coconut dregs (CD) fermented by Saccharomyces cerevisiae for different duration of time on egg production, feed intake, FCR, dry matter digestibility, fecal dry matter and egg quality. Coconut dregs were finely ground and autoclaved. The autoclaved CD was added with 0.2 and 0.4% sodium selenite (SS) as a source of selenium. Distilled water was added to the substrates. The substrate was incubated with S. cerevisiae at room temperature for 5 and 7 days. The incubated substrates were harvested and oven-dried at 50^oC for 2 days. A study was conducted by using 105 18-weeks-old-hens. The hens were fed the experimental diets for 6 months. The experimental diets were: control basal diet (Ctl), basal + 5-days- fermented CD without SS (D5-0SS), basal + 5-days fermented CD with 0.2% SS (D5-0.2SS), basal + 5- days- fermented CD with 0.4% SS (D5-0.4SS), basal + 7-days-fermented CD without SS (D7-0SS), basal + 7-days-fermented CD with 0.2% SS (D7-0.2SS) and basal + 7-days-fermented CD with 0.4% SS (D7-0.4SS). The study used a completely randomized design with 7 treatments and 5 replications. Fermentation could decrease crude fiber and increase the protein content of coconut dregs. The addition of selenium into CD prior to fermentation is able to further decrease crude fiber and increase selenium concentration. Total egg mass, FCR, fecal dry matter, and digestible dry matter intake of hens fed the D5-0.2SS diet were better than those fed the control (Ctl) diet. Feeding the hens with D5-0.2 SS and D7-0.2SS diets could slow down the egg mass loss, compared to eggs of birds fed the control (Ctl) diet (3.19 and 3.23 vs 4.73 g). The addition of selenium in coconut dregs prior to fermentation (D5-0.2 SS, D5-0.4 SS, D7-0.2 SS and D7-0.4 SS ) could minimize the deterioration of the Haugh Unit and yolk index of eggs stored for 28 days at room temperature. In conclusion, Fermentation could improve the nutrient quality of CD and the addition of selenium prior to fermentation could further improve their quality. Fermentation of CD for 5 days with the addition of 0.2% sodium selenite (D5-0.2 SS) could improve egg mass production, FCR, Fecal dry matter, digestible dry matter intake and slow down the deterioration of egg quality.

Keywords: coconut dregs, fermentation, laying hen, Saccharomyces cerevisiae, Selenium

Introduction

Coconut dregs were of low-quality feedstuffs for poultry due to the high concentration of fibrous fraction, the imbalanced amino acids and poor physical properties with low bulk density and high water holding capacity (Sundu et al 2009). A number of technologies have been applied to improve the feeding value of coconut by-products but with limited success. The use of commercial enzymes, reduction of particle size (Sundu et al 2005) and fermentation (Hatta et al 2014) were some efforts that have been practiced. Increasing the production of waste products from agricultural and food industries could lead to environmental problems. Animal industries have been playing a role to minimize waste disposal by utilizing agricultural waste for animal feed. However, the use of agricultural waste without technology intervention could deteriorate the quality of many agricultural wastes, such as cassava pulp (Sugiharto et al 2019), coconut dregs (Sundu et al 2019), feather meal (Pertiwi et al 2017), copra meal (Sundu et al 2005) and shallot waste product (Mozin et al 2015).

Fermentation technology has been used to improve the feeding value of the feedstuffs by allowing the microbes to produce enzymes (Bahri et al 2019), breakdown the fibrous and complex substances and convert inorganic compounds to organic nutrients (Sukaryana et al 2010). Production of microbial protein from non-protein nitrogen in the gut of ruminants due to the presence of microbial rumen could be an indication that microbes have the capacity to utilize and bioconvert minerals into more bio-available nutrients. The same logic could be applied that fermentation through the involvement of microbes is able to convert inorganic and toxic selenium to safe and organic form.

As a crucial trace mineral, selenium is an integral component of glutathione peroxidase (GSH-Px). This enzyme serves as an antioxidant (Han et al 2017), functioning to improve immune status. A number of diseases in poultry such as muscular dystrophy, myopathies, mortality and poor growth have been reported by a number of workers (Nesheim and Scott 1958; Walters and Jensen 1963). Selenium could also increase the production of the egg (Liu et al 2020) and egg quality (Surai 2006). Payne et al (2015) found an increased haugh unit of chicken eggs. This was due to the presence of glutathione peroxidase activity that protects the shell and fluid egg from cellular damage by free radicals and thus maintained egg quality during storage (Gravena et al 2011). A study was conducted to determine the effect of fermentation of selenium-added coconut dregs on egg production, digestibility, fecal dry matter and egg quality during 28 days of storage.

Material and methods

Fermentation procedure

Local coconut dregs were daily collected and oven-dried at 60 ^oC for three consecutive days. The dried coconut dregs were sieved to separate the contaminants such a nutshell and other foreign materials. The coconut dregs were finely ground to 1-2 mm size and used as a fermentation substrate. For fermentation, bakery yeast Saccharomyces cerevisiae (Fermipan ^®) was used. Solid-state fermentation was done by using a method of Jacob and Prema (2006). The fine coconut dregs were autoclaved for 20 minutes at 20 psi. The autoclaved substrate of coconut dregs was cooled at room temperature for 30 minutes. The substrate s were thoroughly mixed without or with 0.2% and 0.4% sodium selenite prior to the addition of distilled water to reach 80% moisture. The mixed substrates were incubated with 0.2% Saccharomyces cerevisiae which is equivalent to 346 CFU/g and put the substrates in the transparent plastic bags for 5 days. Aerobic fermentation was done by making some small holes in the plastic bags. After 5 days of fermentation, the fermented coconut dregs were collected and oven-dried at 50^oC for 48 hours. The dried fermented coconut dregs were stored for proximate analysis (AOAC 1990) and the analysis of selenium (Almeida et al 2015). The fermented coconut dregs were then used as a feed additive.

Experimental Animals and diets

The study used a total of 105 – laying chickens, aged 18 weeks old. The birds were purchased from the local chicken farmers. All the experimental protocols executed in this experiment were approved and monitored by the Animal Ethics Committee at the Animal Husbandry and Fisheries, Faculty, The University of Tadulako, Palu, Indonesia. The 18 weeks-old laying chickens were kept in individual battery cages throughout the study for 6 months. Vaccination of the chickens against New Castle Diseases was conducted by muscular instillation at day 2 and 4 months after arrival by using Vaksimune^®ND B1. The basal layer diet as shown in Table 1 was formulated by using a UFFF Software package (Pesti et al 1986). The experimental layer diets used in this experiment are presented in Table 2. Drinking water and the diets were available at all times. The experimental diet in the feeder was topped up at 07.30 am and 4.00 pm. The drinker was cleaned whenever necessary. The pens and surroundings were regularly cleaned throughout the study.

Parameters measured

A number of variables measured in this study were: total egg mass, hen day, feed intake, feed conversion ratio, egg shape index, yolk color, percentages of albumen, yolk and eggshell, dry matter digestibility, fecal dry matter and digestible dry matter intake, egg mass loss, Haugh unit and yolk index. Digestibility study was done for 7 days by placing a plastic tray underneath each pen. The collection of total feces was carried out at 08.00 am for three consecutive days. The total fecal discharges were weighed after all the contaminated materials such as feed particles and feathers were removed by hand-picking. The samples were oven-dried at 50^oC for three days. The dried fecal samples from each day of the collection were pooled and finely ground. The ground samples were analyzed for the dry matter to measure dry matter digestibility. The dry matter digestibility was determined based on the method of total fecal collection.

Egg collection was done on a daily basis and the eggs were weighed on the same day of collection. Measurements of percentages of albumen, yolk, shell, egg shape index and yolk color were conducted on day 1. The yolk color was measured by using Yolk Color Fan. Two eggs per experimental unit were stored at room temperature for 28 days. Measurement of egg mass reduction was conducted by weighing the same egg on days 1, 14 and 28. Haugh unit and yolk index were measured on days 1, 14 and 28.

Design of experiment and statistical analysis

A Completely Randomized Design with 8 treatment diets and 5 replications (Steel and Torrie 1980) was applied in this trial. Variance analysis was used to determine the significant effect of experimental diets on parameters by using the statistical program of Minitab 16 (Pesti et al 1986). Any differences found in the variance analysis were further tested with the Least Significant Difference test using the Minitab statistical software.

Results

Fermentation of coconut dregs either with or without sodium selenite supplementation produced lower crude fibre and higher protein content than the unfermented coconut dregs (Table 3). Fermentation also leads to biomass loss (Table 3).

The hens fed the D5-0.2SS diet had higher total egg production, egg mass, fecal dry matter and digestible dry matter intake and better FCR than those hens fed the Ctl diet (Table 4). The hens fed D5-0.2SS and D7-0,2SS diets produced lower egg mass loss (Table 5). The hens fed the D5-0.2SS, D5-0.4SS, D7-0.2SS and D7-0.4SS diets had better Haugh unit and yolk index (Table 5).

Discussion

Proximate profiles and biomass loss of fermented coconut dregs

As sodium selenite contains 45.7% selenium, adding 0.2% of this compound in the CD before fermentation was logically able to increase the Se concentration of fermented CD to 914 ppm. Fermentation for 5 and 7 days by Saccharomyces cerevisiae could only yield 720 and 580 ppm Se in the fermented coconut dregs. This is equivalent to between 63 and 79% of total Se present in sodium selenite. This indicates that about 21 to 37% of Se lost during the process of fermentation. The same situation was found when the addition of sodium selenite in the substrate was increased to 0.4%. Fermentation for 5 and 7 days of the 0.4% Se-added substrates lead to 62 and 80% selenium lost respectively. This loss might indicate that the yeast metabolized the inorganic Se via methylation of selenite that could produce hydrogen selenite as a waste product and the gaseous product was freely released into the air. A strong and toxic aroma smelled in this present work might be an indicator of the hydrogen selenite production during fermentation to convert inorganic selenium into seleno-protein. According to Demirci et al (1999), about 0.3% of selenium could be converted into seleno-methionine by Saccharomyces cerevisiae. Since the yeast grew and utilize the inorganic selenium present in the substrate, a longer time of living means more nutrients were used for maintenance and growth. This the reason why 7 days-fermented coconut dregs had a lower selenium concentration.

As a living creature, Saccharomyces cerevisiae needs nutrients to support their growth. The present study indicates that among the major component of nutrients (lipids and crude fiber), Saccharomyces cerevisiae utilized and metabolized more carbohydrates than other nutrients as the crude fiber content drastically dropped. Selenium addition into the coconut dregs prior to fermentation could further decrease the crude fiber concentration. This finding was supported by the previous finding of Hatta et al (2014) and Sundu et al (2019). Protein concentration, on the other hand, increased due to fermentation and selenium addition. The mechanisms might be due to either the conversion of air nitrogen into the protein of yeast or the reduction of crude fibre fraction in the coconut dregs. The reduction in crude fibre or carbohydrate as a source of energy for microbes during fermentation leads to a reduction in its percentage. Accordingly, the percentage of other fractions such as protein increased to meet the total 100% of all proximate fractions.

Effect of fermented coconut dregs on egg production

Studies on the effect of selenium supplementation in the diet on egg production have been reported by many workers (Payne et al 2005; Leeson et al 2008; Suchy et al 2014; Han et al 2017). The findings of the previous studies come up with inconsistent results due to a lot of factors affecting egg production, such as the concentration of Se in the diet and source of selenium (Arpasova et al 2009). A study of Payne et al (2005) indicated that the addition of selenium in the diets, either in the form of sodium selenite or yeast selenium could not enhance egg production while Leeson et al (2008) found that increased Se concentration from 0.1 ppm to 0.3 ppm in the diet enhanced egg production from 64.9 to 72.3%. This current finding adds up to this inconsistency.

In this present study, the addition of 5 days-fermented coconut dregs with the addition of 0.2% sodium selenite as a source of selenium (D5-0.2SS) increased total egg production and egg mass, while other diets containing a high concentration of Se (D5-0.4SS, D7-0.2SS and D7-0.4SS) could not increase total egg production. It is hard to precisely elaborate on this finding. It seems that 5 days of fermentation is the proper duration for incubation time to produce better coconut dregs added with 0.2% sodium selenite. Bahri et al 2020 found that fermentation of coconut dregs for 120 hours (5 days) produced more mannose and enzyme activity than 2, 3, 4 and 6 days fermentation. After 5 days of fermentation, mannose production and enzyme activity dropped drastically. This might be the reason why fermenting coconut dregs for 7 days with the same concentration of selenium could not produce better substrate in the aspect of improving egg production, compared to birds fed the control basal diet. The addition of 0.4% selenium in the substrate might be too much for the improvement of coconut dregs quality, even when the substrate was fermented up to 7 days. Sodium selenite might not be fully converted into organic substances that can enhance egg production.

Effect of fermented coconut dregs on feed intake, digestibility and fecal dry matter

Fermentation has successfully played a role in improving the quality of a number of agricultural by-products such as copra meal (Hatta et al 2014), rice bran and palm kernel cake (Sukaryana et al 2010). Engberg et al (2009) found that fermentation of the diet increased egg production from week 18 to 29. The increase in egg production was due to the fact that fermentation could produce an enzyme (Bahri et al 2019), reduce the concentration of phytate and this could increased the digestibility of protein (Carlson and Poulsen 2003). However, this present study indicates that fermentation of coconut dregs without selenium addition, either for 5 days (D5-0SS) or 7 days (D7-0SS) could not increase the feeding value of the diet as indicated by unchanged total total egg production. The discrepancy of egg production was possible because Engberg et al (2009) fermented the whole diet offered to the laying hens, while this present study used a very small amount of fermented coconut dregs included in the diet. It seems that the quantity of fermented coconut dregs in the diet was too low to boost up the quality of the total diet.

Although, feed intake and dry matter digestibility were not different found in the present study, the accumulated improvement of both variables led to the increase in digestible dry matter intake (DDMI). This becomes one of the reasons for the increased total egg production and egg mass of hens fed the D5-0.2SS diet. Understandably, the amount of digested nutrients absorbed by the hens was used for egg production. This improvement of DDMI might partly be due to the improved health status of the digestive tract of hens fed the D5-0.2SS diet. Read-Snyder et al (2009) found that longer villi of the virus - challenged birds fed the selenium-supplemented diets.

The feed conversion ratio of the control (Ctl) diet was about 2.24. Supplementation of the Ctl diet with 0.5% of 5-days-fermented coconut dregs with the addition of 0.2% sodium selenite (D5-0.2SS) produced better FCR than the control diet. The increase in total egg production of the birds fed the D5-0.2SS diet and the unchanged feed intake led to the improved FCR and thus increased income over feed cost. Comparing to the use of the control diet, application of the D5-0.2SS diet in the formulation of laying hen diets could create economic benefit for the poultry farmers.

Interestingly, fecal dry matter of birds fed the fermented coconut dregs produced drier fecal discharges than the excreta of birds fed the control diet. The addition of the 0.2% selenium into coconut dregs prior to 5 days fermentation(D5-0.2SS) also increased fecal dry matter. Fermentation of coconut waste could produce several enzymes, such as cellulase (Hatta et al 2014) and mannanase (Bahri et al 2019). These two enzymes are responsible for the breakdown of cellulose and mannan that are the major component of fibrous substances in coconut by-products (Sundu et al 2012). Accordingly, the increase in fecal dry matter due to fermentation possibly because these two hydrophilic fibrous components are chopped down into simpler fractions. The finding of decreased excreta dry matter was very important as the issue of higher ammonia production has been an environmental issue in poultry production. However, when the concentration of selenium added in the substrate was increased and the duration of fermentation was extended, the fecal dry matter did not increase, compared to the feces of the controlled birds.

Effect of fermented coconut dregs on egg quality

Although there was an increase in egg mass, the percentage of each ingredient of the egg ( yolk, albumen and eggshell) was unaffected. This might indicate that the proportion of each part of the egg is relatively stable without any impact from the treatment diets. This finding is in accordance with the previous finding of Gjorgovska et al (2012). We found the same quality of yolk and albumen height of the eggs when they were measured on the first day of collection. The logic of this finding is that the height of the albumen and yolk was proportional to the size of the eggs. It can be stated here that the impacts of the external factors such as temperature and duration of storage in this present study were minimal or even non-existent on the first day of storage. The unaffected albumen height was also found in the studies of Han et al (2017) and Liu et al (2020).

Studies on the effect of supplemental selenium in the diet of laying hens on yolk color and egg shape index have been done by a number of authors (Renema and Sefton 2004; Arpasova et al 2009; Han et al 2017; Liu et al 2020). The authors found a non-significant difference in yolk color (Han et al 2017; Arpasova et al 2009) and egg shape index (Liu et al 2020) due to supplementation of the diet with selenium. Since yolk colour was affected by beta carotene, the addition of selenium prior to fermentation of coconut dregs might not increase beta carotene absorption in the digestive tract of birds and thus could not affect yolk colour. This present study supports and adds up the consistency of the previous findings.

The effects of supplemental Se in the poultry diet on egg mass loss, HU and yolk index have been studied by Payne et al 2005, Pappas et al 2005, Gravena et al 2011 and Han et al 2017. Since eggs are a perishable product, containing a high quantity of protein and lipid, this product undergoes physical, biological and chemical changes during storage, particularly when storage temperature was not set up properly. This current finding indicated that when the egg was stored for 14 days at room temperature, the egg mass lost between 1.82 to 2.58%. The drastic loss took place on eggs produced by hens fed the control (Ctl) diet when the eggs were kept 28 days. The supplementation of the diets with the 0.2% additional selenium fermented for 5 days (D5-0.2SS) and 7 days (D7-0.2SS) could slow down the egg mass loss, compared to eggs from hens without selenium supplementation in the diet. Since eggshell thickness, porosity and size of egg are the factors affecting egg mass loss, it seems that selenium on the right concentration might produce better eggshell porosity and thicker eggshell. This condition can slow down the evaporation of water from albumen or yolk. A study by Nemati et al (2020) indicated that selenium could increased the thicknees of eggshell. The less weight loss of egg produced by hens fed the D5-0.2SS and D7-0.2SS means less revenue loss and accordingly, this condition can affect the total income.

It has been believed that the Haugh unit was affected by selenium diets (Pappas et al 2005) duration of storage and temperature of storage (Gravena et al 2011). The effect of selenium on HU becomes evident when the eggs were stored for a couple of weeks. A study of Gravena et al (2011) indicated that when the quail egg was stored for 30 days, the Haugh unit dropped dramatically from 89.6 to 71.1. The authors kept the eggs in the refrigerator and found a small reduction in HU from 89.6 to 87.4, compared to the reduction from 89.6 to 78.9 of the eggs kept at room temperature. The present study indicated that there was no significant effect of supplemental selenium in the diets when the eggs were stored for only 14 days at room temperature. However, when the length of storage was extended to 28 days, HU of eggs from hens fed the control Se-unsupplemented diet, dropped drastically to 47.7. The eggs produced by hens fed the D5-0.2SS, D5-0.4SS, D7-0.2SS and D7-0.4SS (selenium-supplemented diets) were 69.6, 69.7, 69.8 and 68.0 respectively. Fermentation of coconut dregs without the addition of Se (D5-0SS and D7-0SS) could not maintain the HU score when the eggs were stored for 28 days.

The same trend was also found in the yolk index parameter in which supplementation of the diet with selenium could slow down the deterioration of the yolk index during storage. This finding was supported by the previous finding of Gravena et al (2011). Since fermentation could not maintain the quality of yolk when the eggs were kept for 28 days at room temperature, it is most likely that the factor impacting the yolk quality during storage was selenium concentration in the eggs. A previous finding of Leeson et al (2008) indicated that hens fed the yeast selenium – supplemented diets produced more selenium in the eggs. It has been well accepted that selenium plays a role in the production and activity of glutathione peroxidase (GSH-Px). A better yolk index of birds fed the selenium-supplemented diets (D5-0.2SS, D5-0.4SS, D7-0.2SS and D7-0.4SS ) is possibly due to the production and activity of GSH-Px as an antioxidant enzyme functioning to maintain the integrity of the cell and antioxidative protection from the oxidation of yolk lipid. Measurement of thiobarbituric acid reactive substances (TBARS) to determine fatty acid peroxidation of yolk lipid was done by Gajcevic et al (2009). The authors found that the TBARS yolk lipid concentration as an indicator of lipid peroxidation was lower in the egg of hens fed the higher selenium diet when the eggs were kept for 28 days, but not for 14 days storage.

Conclusions

• Fermentation could improve the nutrients quality of coconut dregs
  
  
• Improvement of total egg production, egg mass, FCR, fecal dry matter, and digestible dry matter intake was reached due to the addition of diets containing the 5 days-fermented coconut dregs added with 0.2% selenium (D5-0.2SS)
  
  
• Selenium-added diets (D5-0.2SS, D5-0.4SS, D7-0.2SS and D7-0.4SS ) could minimize the deterioration of the Haugh unit and yolk index of eggs stored for 28 days at room temperature

Acknowledgment

Our profound thanks firstly go to the Ministry of Research, Technology and Higher Education of the Republic of Indonesia for the provision of financial support, enabling us to carry out this study. We are indebted to The University of Tadulako, Faculty of Animal Husbandry and Fisheries for its research facilities. Our students for their work in collecting the coconut dregs, doing fermentation and taking care of the hens are all appreciated.

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