Effects of processing protocols on the potential of hatchery waste as a protein source for livestock production

Asiedu P, Odoi F N A², Prah A A², Wallace P Y², E K Adu¹, Bumbie G Z and Osei D Y¹

University of Energy and Natural Resources, Box 214 Sunyani, Dormaa Campus
peter.asiedu@uenr.edu.gh
¹ Council for Scientific and Industrial Research, Animal Research Institute, Achimota, Accra, Ghana
² Department of Animal Science, School of Agriculture, University of Cape Coast

Abstract

Raw hatchery waste comprising of infertile eggs, egg shells, dead-in-shells and low-grade unsalable chicks, was subjected to three different processing protocols to determine the potential as a protein source for livestock production. The hatchery waste was first sorted into the various components and sterilized by steaming and drying at different temperatures resulting in nine different protocols: steaming for 5 min and drying at 60^oC (5M60T), steaming for 5 min and drying at 70^oC (5M70T), steaming for 5 min and drying at 80^oC (5M80T), steaming for 10 min and drying at 60^o C (10M60T), steaming for 10 min and drying at 70^oC (10M70T), steaming for 10 min and drying at 80^oC (10M80T), steaming for 15 min and drying at 60^oC (15M60T), steaming for 15 min and drying at 80^oC (15M70T), and steaming for 15 min and drying at 80^oC. Samples of each treatment were subjected to proximate analyses and microbial evaluation. The results of the proximate analysis show that the hatchery waste meal (HWM) is rich in protein, ash, dry matter and fat. The percentage residual crude protein (CP) content in the differently processed HWM samples were 38.57% (5M60T), 44.71% (5M70T),49.90% (5M80T), 38.41% (10M60T), 46.24% (10M70T), 48.82% (10M80T), 38.26% (15M60T), 45.63% (15M70T) and 47.81% (15M80T) respectively. Again, the mean total viable counts for 5M80T, 10M60T, 10M80T, 15M60T, 5M70T and 15M80T were 9.30, 9.80, 9.54, 9.40, 9.95 and 9.98 (log/cfu/g) respectively. In conclusion, it can be inferred that hatchery waste meal processed at 5M80T appears to have the best bacterial load reduction effect as well as high crude protein level.

Keywords: dead-in-shells, egg shells, infertile eggs, low-grade unsalable chicks and hatchery waste meal

Introduction

The Ghanaian livestock industry is larger, more concentrated, and more technically advanced than it was two decades ago. The world is facing malnutrition and as a result, the traditional use of maize, wheat and soybean in the livestock feed can be substituted with newly developed livestock feed, which is usually a by-product of animal waste. The current trend toward animal wastes recycling is motivated by both economic and environmental considerations. Since feed cost is about 70 percent of the total animal production costs, the substitution of conventional feed by processed animal waste will lead to a significant reduction in the costs of animal feed and the ultimate derived products (Sung and Kim 2019).

One such unorthodox feed is the hatchery waste (Mahmud et al 2015 cited by Djang-Fourdour 2016). The poultry industry everywhere in the world produces quiet a significant amount of hatchery waste (Djang-Fourdour 2016). It includes egg shells, infertile eggs, dead embryos in the shell and dead chicks. Hatchery waste can be converted into nutritionally dense meal through proper processing.

The disposal of hatchery waste (HW) is of great concern to the poultry industry, as well as the general public in many countries, including Ghana. The only concern in the new feed is its microbial count, the problem of rancidity due to formation of free fatty acids and the variability of its quality (Glatz et al 2011).

Worldwide, the common ways of disposal are incineration, rendering method as well as land fill (Shao et al 2008). This makes the disposal of HW a very expensive venture to producers and, also unsafe to the environment in general (Shao et al 2008). Hatchery waste when managed and processed appropriately, the waste material has the potential of increasing viable economic profitability of the poultry industry (Das et al 2002 and Adeniji and Adesiyan 2007). The poultry industry will have to develop innovative technologies and techniques that may benefit it economically and environmentally. For these reasons, the objectives of the present study were to determine the nutritional data of hatchery waste meal (HWM) processed using different techniques as potential animal protein source for livestock.

Materials and methods

Study Area

The study was conducted at the Council for Scientific and Industrial Research-Animal Research Institute (CSIR-ARI) located in the Adentan Municipal District (5°42′25″N 0°10′15″W), in the Greater-Accra Region of Ghana. Nine categories of hatchery waste samples were collected for proximate and microbiological analyses.

Collection and processing of hatchery waste

Hatchery waste (HW) was obtained from the CSIR-Animal Research Institute’s hatchery at Katamanso and a private hatchery at Kodiabe, Accra. The raw hatchery waste comprised of infertile eggs, egg shells, dead-in-shells and low-grade unsalable chicks. Equal quantities of 3 kg of raw mixture of HW were placed in three different polythene bags of different colours. The polythene bags containing the HW were then immersed in a steel drum containing boiling water. The steel drum together with its contents were subjected to a constant boiling temperature of 100 ^oC for 5, 10 or 15 minutes. The steamed samples were removed from the drum and the sample pulverized using a wooden mortar and pestle. This was to reduce the particle size of the hard tissue and facilitate drying.

Samples from all the three different steaming procedures were then dried in a hot air oven set to three different temperature regimes i.e steaming for 5 min and drying at 60^oC (5M60T), steaming for 5 min and drying at 70^oC (5M70T), steaming for 5 min and drying at 80^oC (5M80T), steaming for 10 min and drying at 60^oC (10M60T), steaming for 10 min and drying at 70^oC (10M70T), steaming for 10 min and drying at 80^oC (10M80T), steaming for 15 min and drying at 60^oC (15M60T), steaming for 15 min and drying at 80^oC (15M70T), and steaming for 15 min and drying at 80^oC (15M80T).

The samples were then, ground in a JF automated machine using initially an 8 mm dial screen and subsequently, a 2 mm dial screen in a hammer mill. Representative samples of the HWM prepared from the 9 previously described processing techniques were then taken, sub-divided into three (3).and placed into tared, zipper-locking plastic bags and weighed. All sub-samples were subjected to proximate analyses (AOAC 2005) and microbial analyses (Deere et al 2002).

Shows a pictorial representation of how the various processes of raw HWM were taken through to obtain a form in which it could be incorporated into diets. Figure 1.

Figure 1. Steps involved in processing raw HW into HWM (hatchery waste meal)

Chemical analysis

All chemicals used during the study were of analytical grade. The dry matter content, ash, crude protein and ether extract in the processed HWM samples were determined using standard methods, as described AOAC (2000). All analyses were performed in triplicate and results presented as mean values ± SD.

Microbial analysis

Sample Preparation

From each of the nine treatment specimens, 1 g was aseptically transferred into sterile MacCartney bottles containing 9 ml of 0.1% sterile blank peptone water [Merck, Darmstadt-Germany] to form the neat. These suspensions were incubated at 37 ⁰C for 10 - 15 minutes in a Wagtech bacteriological incubator.

Media preparation

Media such as Membrane Lactose Glucorunide Agar (MLGA), [Oxoid, CM 1031 Hamsphire – England], Standard Plate Count Agar (SPCA) [Merck, Darmstadt-Germany], Xylose Lysine Desoxycholate (XLD) [Oxoid, CM 0469 Hamsphire – England] and blood agar were used for the microbial assay. These media which were in powder form(s) were reconstituted with distilled water according to manufacturer’s instructions. The required quantities of agar were weighed into conical flasks and dissolved by adding distilled water. They were sterilized by autoclaving at different temperatures as stated by manufacturer prior to use.

Counting of microorganisms

Bacterial enumeration was done using pour-plate and plate - count techniques. Samples were serially diluted using 10-fold serial dilution into nine (9) other sterile MacCartney bottles containing 0.1% 9 ml peptone water. Different pipette tips were used for each dilution.

Total viable count

For total aerobic mesophilic bacteria (TAMB) count, the pour - plate method was used. One (1) ml of each dilution was aseptically added to 9 ml of molten Standard Plate Count Agar [Merck, Darmstadt-Germany] kept at 45-50 ⁰C in a water bath [Grant, OLS 200]. This was mixed by rotation and poured into 9 cm sterile petri dish. It was allowed to set and incubated at 37 ⁰C for 18-24 hrs. After incubation, plates showing collonial growth were selected and counted using electronic colony counter [Stuart Scientific]. Colonies counted for each plate was multiplied by the dilution factor to obtain the number of colonies.

Total coliform count

Using the plate-count method, one (1) ml of each dilution was aseptically put into 9 cm petri dish. Nine (9) ml of molten Membrane Lactose Glucuronide Agar (MLGA) [Oxoid, CM 1031 Hamsphire – England] kept at 45-50 ⁰C in a water bath was added, mixed by swirling and allowed to set. Plates were incubated at 37 ⁰C for 24-48 hrs and examined for colonial growth.

E. coli count

One (1) ml of each dilution was aseptically put into 9 cm petri dish using the plate-count technique. Into same plate, nine (9) ml of molten Membrane Lactose Glucuronide Agar (MLGA) [Oxoid, CM 1031 Hamsphire – England] and kept at 45-50 ⁰C in a water bath was added. This was mixed by swirling and allowed to set after which plates were incubated at 37 ⁰C for 24-48 hrs.

Salmonella count

One (1) ml of the neat sample was added to 10ml of double strength Selenite F broth [Oxoid, CM 395 Hamsphire – England] for enrichment. This was mixed thoroughly and incubated at 37 ⁰C overnight. After incubation, 1 ml of the culture (SF broth) was serially diluted using 10-fold serial dilution into nine (9) other sterile MacCartney bottles containing 0.1 % 9 ml peptone water. Using pour-plate technique, 1 ml of diluent was aseptically added to 9 ml of molten Xylose Lysine Deoxycholate (XLD) agar [Oxoid, CM 469, Hamsphire – England] and kept at 45-50 ⁰C in a water bath. It was mixed by rotation and incubated at 37 ⁰C for 24 hrs.

Culture Techniques

Using a sterile inoculating loop, the neat samples were plated-out onto blood agar [Merck, Darmstadt-Germany] and xylose lysine desoxycholate (XLD) agar [Oxoid, CM 0469, Hamsphire – England]. Plates were incubated aerobically at 37 ˚C for 24-48 hrs in a bacteriological incubator (Plate 4). Cultures were examined for colonial characteristics on the media. Impure cultures on primary media were purified by subculturing onto selected secondary media to obtain discrete colonies.

Isolation and identification of organisms

After overnight incubation, colonial morphology of organisms based on their physical characteristics was studied for size, shape, outline, colour and change in medium on various media. Standard microbiological techniques including Gram staining, cellular morphology [of organisms using compound microscope magnified at x100 with oil immersion] and biochemical tests such as Motility Indole Urea (MIU) [Lioflichems.r.l. Bacteriology Products, 610236, Italy], Catalase, Triple Sugar Iron (TSI) [Oxoid, CM 0277, Hamsphire – England], Indole Methyl Red Vorges-Proskeur Citrate [IMViC] test, carbohydrates Oxidation/Fermentation (O/F) test [to detect gas and or acid production] among others were applied to isolate and identify the organisms.

Culture, Isolation and Identification of Salmonella

Using the plate-out technique, subcultures were made from the Selenite F broth aseptically onto XLD agar [Oxoid, CM 469, Hamsphire– England]. Cultures were incubated at 37⁰C for 24 -48hrs and examined for the physical characteristics of colonies on the media.

Culture, Isolation and Identification of Escherichia coli

All lactose fermenting colonies on MacConkey agar were selected and aseptically subcultured onto MLGA [Oxoid, CM 1031 Hamsphire – England] to isolate and identify E. coli. Cultures were incubated at 45 ⁰C for 24-48 hours in a bacteriological incubator.

Statistical analyses

The data obtained were subjected to analysis of variance (ANOVA) and the means of the treatments compared statistically using the Tukey’s test at 95% confidence level. The count obtained (cfu g⁻¹) for the microbial analysis were first converted to logarithmic form. The logarithmic was compared with the isolate identified according to relative linearity based on Statistical analysis of both results by using Microsoft Excel Statistics 2002 for Windows. Differences in results that exceeded 1 log CFU g⁻¹ were classified as discrepancies (Hutton, 2001).

Results

Proximate Composition of Processed HWM

Proximate compositions of samples of HWM processed using different processing protocols or techniques are presented in Table 1. The results of the proximate analysis show that all the HWM samples were rich in protein, ash, dry matter and fat. The protocol used in this study again indicate that crude protein (CP) content of the HWM varied depending on the drying temperatures and periods of exposure. The 5M60T, 5M70T, 5M80T, 10M60T, 10M70T, 10M80T, 15M60T, 15M70T and 15M80T HWM registered CP levels of 38.57, 44.71, 49.90, 38.41, 46.24, 48.82, 38.26, 45.63 and 47.81 respectively. Dry matter was similar for 5M60T, 5M80T, 10M80T and 15M80T and significantly (p>0.05) higher than the rest of the treatment. Numerically the highest value for DM was observed on treatment 5M80T. However, the highest Ash content value recorded in treatment 5M80T was (33.46 %), followed closely by 15M70T (32.05 %) while the least was found in 15M60T (29.56 %). With respect to ether extract content, 10M70T had statistically highest EE content (23.52 %) value while 5M80T had the least (17.72 %).

Comparison of Mean Bacterial Loads

The study was undertaken to evaluate microbial quality of HWM before storage and on day 90 in other to test for the presence of Escherichia coli, Streptococci, Salmonella and Lactobacillus (AOAC 2000). Table 2 and 3 compares the mean bacterial loads on HWM from the nine different processing protocols. The mean total viable counts (Table 2) for 5M60T, 5M70T and 10M70T were 10.2, 10.5 and 10.2 respectively, and were higher than the accepted values of 6.0 (log) (cfu/g) according to Microbiological Specification for Food ICMSF (1998). On the contrary, the mean total viable count (Table 3) for 5M60T, 5M70T and 10M70T were 8.7, 8.0 and 8.1 respectively and were within the range, recommended by ICMSF (1998). Again, the mean total viable counts (Table 2) for 5M80T, 10M60T, 10M80T, 15M60T, 15M70T and 15M80T were 9.30, 9.80, 9.54, 9.40, 9.95 and 9.98 respectively and were within the range, recommended by the Hutton (2001).

The importance of temperature for bacterial growth can be assessed at different critical points between processing and consumption of a product in particular during sample handling and storage of the product. The results from this study show that all the HWM samples from the 9 processed HWM were contaminated with some level of microbes of different genera (Table 3 and 4). The major contaminants were Gram-positive bacteria.

Discussion

Chemical compositions of hatchery waste meal are highly variable (Sung et al 2019) which is likely due to variation in the ratios of hatchery waste ingredients and processing methods. The highest crude protein content in the hatchery waste meal samples (49.90 %) was similar to values reported for hatchery waste of 48.25% by Glatz and Miao (2009), but higher than 44.3% reported by (Rasool et al 1999). A report by Khan et al (2005) and Abiola et al (2012) recorded crude protein content of 42.26% for HWM. These levels of CP suggest that HWM is likely to be highly nutritious and good for use in livestock feed, including those for pigs. There are indications that factors which affect crude protein content in HWM are the proportion of egg shells processing technique (particularly the temperature) and treatment period (Khan and Bhatti 2002). This is in support of the findings of Rasool (1999) and Shahriar et al (2008) that HWM has good nutritional attributes with the potential to be used as livestock feed. Abiola (2010) indicated that HWM is highly nutritious and compared favourably with fish meal, and could be a good supplement for cereals or carbohydrate meals. However, while it is rich in calcium, it is low in phosphorus. Calcium level was variable and depended on the presence of shell moiety and hatch percentage. Schaafsma et al (2002) reported a positive effect of egg shell calcium supplementation (with added magnesium and vitamin D) on bone mineral density (BMD).

The ether extract content of 17.72% was lower than the 20.28% reported by Aydin and Gumus (2012), but higher than the report of Abiola et al (2010) who obtained a value 5.45%. The higher ether extract content of HWM in this study could be attributed to a higher egg yolk content of infertile eggs in the processed samples. The average ash content of 27.09-33.46% which could be due to the high content of eggshell at the time of processing. AFRIS (2007) indicated that approximately 84% of the egg shell is ash, of which most is calcium carbonate. Before conducting the present study, microbial analyses on hatchery waste ingredients were performed resulting in no evidence of spoilage (Lee et al 2018).

In the present study, the processing techniques did not eliminate the viable counts of bacteria completely but managed them to safe levels. All 3 types of processing techniques were found effective in counter acting TCC, as there were no significant (p > 0.05) differences in TCC levels in the processed meals. Hatchery waste meal processed for 5 mins at 80^oC appears to have the best bacterial load reduction effect. According to USDA (2006), micro-organisms can be found on the outside and inside of the egg shell. The bacteria genera that were isolated were identified as Corynebacterium glutamicum, Corynebacterium diphtheria, Corynebacterium spp, Bacillus cereus and Staph. aureus . There was no isolation of enterobacteria species. Results of the present study are in line with findings of Osei-Somuah et al (2003), who determined total number of aerobic micro-organisms present in unextruded poultry by-product meal diets and reported levels of 47000 cfu/g which could be completely eliminated by a high temperature and short time extrusion process. According to Miller (1984) when hatchery wastes were processed through high temperature extrusion, no Salmonella organisms were found.

In a similar study, Dhaliwal et al (1998) concluded that Bacillus and Streptococcus species in raw HW could be eliminated after processing with extrusion. These also agreed with Osei-Somuah et al (2003) who isolated and identified similar microorganisms during work in the southern part of Ghana, confirming that these organisms can survive under different temperature conditions (i.e. from 4 °C to 60°C). However, Salmonella spp., a common pathogen of poultry was not isolated in this study suggesting that the organism could not survive the temperatures the HMW were subjected to. The presence of environmental organisms such as Bacillus, Staphyloccocus and Corynebacterium spp. in HWM is an indication that these microbes were isolated from the shell surfaces of eggs sampled and also possibly from the deep litter system where eggs are laid on the floor.

Conclusions

• Hatchery waste meal was observed to compared favorably with protein level in fish meal (49.90 Vrs 52.04 %).
  
  
• The study again showed that HWM after simple processing (steaming) ended up without much loss in protein content (5M80T).
  
  
• Hatchery waste meal processed at 5M80T appeared to have the best bacterial load reduction effect as well as highest crude protein level.

Acknowledgements

The author would like to thank the immediate past Director, Prof. E. K. Adu of the CSIR - Animal Research Institute and the Animal Science Department, UCC and the staff of the Quality Control and Microbial Laboratories of the CSIR-Animal Research Institute for their technical support.

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