Effect of different levels of molasses, salt and antimycotic agents on microbial profiles during fermentation of poultry intestine

Effect of different levels of molasses, salt and antimycotic agents on microbial profiles during fermentation of poultry intestine

Bioresource Technology 63 (1998) 237-241 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER Pli:S...

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Bioresource Technology 63 (1998) 237-241 © 1998 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0960-8524/98 $19.00 ELSEVIER

Pli:S0960-8524(97)00136-3

EFFECT OF DIFFERENT LEVELS OF MOLASSES, SALT AND ANTIMYCOTIC AGENTS ON MICROBIAL PROFILES DURING FERMENTATION OF POULTRY INTESTINE D. M. Shaw, D. Narasimha Rao & N. S. Mahendrakar* Meat, Fish and Poultry Technology, Central Food Technological Research Institute, Mysore-570 013, India (Received 10 August 1997; revised version received 2 September 1997; accepted 8 September 1997) molasses (Durairaj et al., 1976; Urlings et al., 1993; Zuberi et al., 1993; Levin, 1994) have been used as a source of fermentable sugars to ensure successful preservation. In order to develop an appropriate fermentation ensiling technique, the work was designed to determine the optimum level of molasses as a source of carbohydrate, with common salt, to see its effect on microbial profile during fermentation of offal. Ethoxyquin at 0.02% (w/w) was added to the fermentation mixture as it was found to be effective in reducing oxidative rancidity (Mahendrakar et al., 1995).

Abstract

Waste poultry intestines (PI) consist of 75.8% water, 7.9% ether extract, 12.4% protein and 1.7% ash. The process of ensiling PI after mixing with different levels of molasses (5-12% w/w) and common salt (0.5-2.0% w/w) was studied under microaerophilic conditions at Indian ambient temperature (26-t-2°C). The inhibitive effects of benzoic, propionic and sorbic acids on growth of yeasts and moulds during fermentation was also studied. The results showed that molasses at less than 10% was inadequate and at 12% had no additional advantage for effecting fermentation. Salt at 0.5-2.0% (w/w) had no marked effect on pH and the microbial profile. Propionic acid at 0.5% (v/w) was quite effective in controlling the growth of yeasts and moulds. A microbiologicaUy safe silage product can be prepared by homogenising PI with 10% (w/w) molasses and 0.5% (v/w) propionic acid and fermenting under microaerophilic conditions at ambient temperature (26+_2°C) for 6days. © 1998 Elsevier Science Ltd. All rights reserved.

METHODS

The intestines of poultry (PI) slaughtered and dressed in the local market were collected in a batch of 8-10 kg in a plastic bucket, brought to the laboratory, homogenised to a pasty consistency using a Stephan Universal Machine (Stephan Food Processing Technology, Germany), divided into 2 kg portions and treated as follows:

Key words: Poultry intestines, fermentation, ensilage, molasses, antimycotic agents, microbial profile.

Experiment I: 5, 8, 10 and 12% (w/w) of molasses. Experiment II: 10% of molasses and 0.5, 1.0 or 2.0% of common salt. Experiment IIh 10% molasses and 0.1, 0.3 and 0.5% of benzoic(w/w), propionic (v/w) or sorbic (w/w) acids.

INTRODUCTION

Annually, India produces about 65000 MT (metric tonnes) of poultry intestines (PI) from slaughter and dressing of poultry (Anon, 1995). At present this offal is rejected, causing environmental pollution besides loss of possible nutrients. Fermentative ensiling of this offal could be an economical process to prepare a product for use as an ingredient in animal feeds as an alternative to the conventionally used fish meal, which is expensive and is limited in supply by the availability of the raw material. Animal offals are poor in carbohydrates. Fermentation can be effected in offals with addition of fermentable sugars. Corn syrup (Levin, 1994) or

The molasses used was of grade II, as described previously (Ahmed and Mahendrakar, 1995). The treated homogenate (2 kg) was transferred into a 3 1 beaker, layered with a thin, low density, polyethylene film and the beaker covered with a lid. The mixture under such a microaerophilic condition was allowed to ferment at ambient temperature (26_+2°C) for 6 days. Changes in pH and microbial population of PI during fermentation were monitored periodically, as in the procedures outlined earlier (Ahmed et al., 1996). However, salmonella in PI and in silage

*Author to whom correspondence should be addressed. 237

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D. M. Shaw, D. N. Rao, N. S. Mahendrakar

prepared by fermenting with 10% (w/w) molasses and 0.5% (v/w) propionic acid after attaining pH 4.2 were enumerated by the three tube MPN procedure (APHA, 1984). Proximate composition of PI was determined according to AOAC (1990) procedures. Statistical analysis

Each set of experiments (I, II, III) was repeated six times (six batches of PI). The results obtained were subjected to analysis of variance. Significant differences within means were tested by Duncans multiple range test (Duncan, 1955). RESULTS AND DISCUSSION The analysis of PI used here is shown in Table 1. The intestines harboured large numbers of microorganisms, including spoilage and pathogenic organisms, with quite a large number of coliforms, of which E. Coli formed the major constituent, followed by enterococci and staphylococci (Table 1). A similar observation was also made by Barnes et al. (1971) and Fuller (1973). Approximately 8 log cfu g-1 of yeast and moulds were present in PI. Large numbers of lactic acid bacteria present in the offal would have helped to regulate the intestinal microflora and hence there is a possibility of fermentative ensiling of PI without the addition of a starter culture. Table I. Proximate composition and microbial population of poultry intestine

Proximate composition (g 100 g - ' ) Water Ether extract Protein Ash

Microbial population (log cfu g-1) Standard plate count Coliforms

75.8 7.9 12.4 1.7

7.9 7.9 7.7 6.4 7.4 7.7 8.2

E. coli

Staphylococci Enterococci Yeast and mould Lactic acid bacteria

Each value is a mean of 6 experiments.

Experiment I. Effect of molasses

The mean initial pH of PI homogenate was 6.0 and that of molasses 4.6. After mixing the two, the pH of the mixture was in the range of 5.0-5.3, depending on the level of molasses added. During fermentation, a fall in pH was noticed in all the treatments (Fig. 1). The desired pH of 4.2 (Raa and Gildberg, 1982) was attained in 2 days with 8% molasses and in 1 day with 10 or 12% molasses, followed by a marginal decrease in pH up to 6 days fermentation. On the other hand, with 5% molasses the lowest pH attained was 4.3 at 1-2days, but this slowly increased up to 6 days fermentation. Five percent molasses was inadequate to effect good fermentation and 8% molasses was slower (2 days) than 10% molasses (1 day). Molasses at 12% level had no additional advantage. Fall in pH of fish during fermentation is well documented and reviewed by Raa and Gildberg (1982) and Levin (1994). Similar observations were made in this laboratory when fish viscera were fermented with different levels of molasses (Ahmed and Mahendrakar, 1995). Urlings et al. (1993) also reported similar results during fermentation of broiler processing waste. With regards to the microbial profile of PI during fermentation, the molasses levels had marginal (P > 0.05) effects on standard plate counts and lactic acid bacteria (Fig. 2). Coliforms were eliminated in 3 days and 5 days with 10 and 12% molasses, respectively, but with 5 and 8% molasses coliforms were not eliminated even after 6 days fermentation. E. Coli were eliminated in 5, 4, 3 and 4 days with 5, 8, 10 and 12% molasses, respectively. Counts of staphylococci were generally lower with higher levels of molasses. Enterococci were not eliminated with 5% molasses, but were eliminated in 4 days in all other treatments. However, the high initial yeast and mould counts (8.2-8.6 log cfu g - l ) decreased only slowly, by 1-2 log cfu g-1, after 6 days fermentation. These results again indicated that 10% molasses was optimum to effect good fermentation. Hence in the subsequent experiments, molasses at 10% of PI was used. Experiment II. Effect of salt

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The inclusion of salt at 0.5, 1.0 and 2.0% (w/w) of PI had marginal (P > 0.05) influence on pH (Fig. 3), although slightly lower pH values were observed with higher salt content. The desired pH of 4.2 was observed after i day fermentation of all the samples. The inclusion of salt at these levels generally had a marginal (P > 0.05) effect on the microbial profile of PI during fermentation (Fig. 4). Coliforms and E. coli were eliminated in 3 days and enterococci in 6 days in all the samples. Stapylococci, having a limiting pH of 4.0 (ICMSF, 1980), remained in the silage, but the colonies did not have lipolytic zones. They were also coagulase negative and hence non-pathogenic (APHA, 1984).

Ensiling of poultry entrails

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Staph. aureus counts :only in excess of 6 log cfu g produce sufficient toxin to cause gastroenteritis (Evans, 1986). Salmonella counts in PI were in the range of 2.0-3.1 log cfu g-1 and in the silage with pH of 4.2 or below they were absent, as their limiting pH is 4.5 (ICMSF, 1980). Two and 4% salt had a marked effect (P<0.05) on pH (Ahmed and Mahendrakar, 1995) and resulted in a marginally (P > 0.05) lower number of

all organisms except yeasts, moulds and lactic acid bacteria (Ahmed and Mahendrakar, 1995) during fermentation of fish viscera. The inclusion of salt at 0.5-2.0% (w/w) on wet weight basis corresponds to about 2-8% (w/w) on dry weight basis. Such a high concentration of salt restricts the utility of fermented silage product as an ingredient in animal diet. Inclusion of salt at these levels had no marked effect on microbial population of PI during fermentation. Hence salt was not added to the fermentation mixture in the subsequent experiments. Yeast and mould counts were quite high in all the salted samples as also in the unsalted samples as mentioned previously. In order to study the effect of different antimycotic agents to control the growth of yeasts and moulds, Experiment III was carried out. Experiment III. Effect of antimycotic agents The inclusion of sorbic, propionic or benzoic (at 0.1, 0.3 or 0.5% levels) acids did not influence the pH of the fermentation mixture, although propionic acid produced a slightly lower pH (Fig. 5). The yeast and mould counts decreased during fermentation up to 6 days; approx, to log 4.6-5.1 cfu g - 1 with sorbic acid, log 5.2-5.4 cfu g - 1 with propionic acid and log 6.4-6.5 cfu g - I with benzoic acid, indicating that

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D. M. Shaw, D. N. Rao, N. S. Mahendrakar

During acid silaging of poultry and fish offals, propionic acid was effective in suppressing yeasts and moulds (Mahendrakar et al., 1991). Sorbic acid (0.1%) inhibited growth of yeasts during the initial fermentation silaging of fish/fish waste (Lindgren and Pleje, 1983). In conclusion, a microbiologically safe silage product can be prepared by homogenising poultry intestine with 10% (w/w) molasses and 0.5% (v/w) propionic acid and fermenting the mixture for 6 days

sorbic acid was the most and benzoic acid the least effective (Fig. 6). Between sorbic and propionic acids, the differences were marginal (less than log 1 cfu g - l ) . Sorbic acid is more expensive (approx. 1500 rupees per kg) than propionic acid (approx. 300 rupees per litre) and the latter is more effective in reducing pH (Fig. 5). Considering these advantages, it would be preferable to use propionic acid at 0.5% (v/w) level in offal silage preparation to suppress the growth of yeasts and moulds. lO

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Ensiling of poultry entrails

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The work was supported by a grant from the D e p a r t m e n t of Biotechnology, Govt of India. D.M.S. thanks the Council of Scientific and Industrial Research, New Delhi, for the award of a fellowship. The authors express thanks to the Director of the Institute for extending facilities to carry out the work. REFERENCES

Ahmed, J. & Mahendrakar, N. S. (1995). Effect of different levels of molasses and salt on acid production and volume of fermenting mass during ensiling of tropical fresh-water fish viscera. J. Food Sci. Technol., 32, 115-118. Ahmed, J., Ramesh, B. S. & Mahendrakar, N. S. (1996). Changes in microbial population during fermentation of tropical fresh water fish viscera. J. Appl. Bacteriol., 80, 153-156. Anon (1995). Final report of research project entitled, 'Development of technology for utilisation of food processing wastes and low cost byproducts as a source of protein in preparation of aquaculture feeds' (Nr. GAP-0084). Grant-in-aid by Dept. of Biotechnology, Ministry of Science and Technology, Govt. of India. AOAC (1990). Official Methods of Analysis, 15th edn. Association of Official Analytical Chemists, Washington, D.C. APHA (1984). Compendium of Methods for the Microbiological Examination of Foods. American Public Health Association, Washington, D.C. Barnes, E. M., Mead, G. C. & Barnum, D. A. (1971). The intestinal flora of the chicken in the period 2 to 6 weeks

of age, with particular reference to the anaerobic bacteria. British Poult. Sci., 11, 311-326. Duncan, D. B. (1955). Multiple range and multiple F-test. Biometrics, 11, 1-42. Durairaj, A. S., Santhanaraj, T., Sultan, K. M. & Dorai Rajah, K. A. P. A. (1976). Utilisation of trash fish. Fish silage - - some aspects of processing and storage. In Proc. Symp. Fish Process. Ind., Central Food Technol. Res. Inst., Mysore, India. Evans, J. B. (1986). Staphylococci. In Advances in Meat Research, Vol. 2, 'Meat and Poultry Microbiology', eds A. M. Pearson and T. R. Dutson. AVI, Connecticut. Fuller, R. (1973). Ecological studies on the lactobacillus flora associated with crop epithelium in the fowl. J. Appl. Bacteriol., 36, 131-139. ICMSF (1980). Microbial Ecology of Foods, Vol. 1. Factors affecting life and death of microorganisms, pp. 92-110. International Commission on Microbiological Specifications for Foods. Academic Press, London. Levin, R. E. (1994). Lactic acid and propionic acid fermentation of fish hydrolysates. In Fisheries Processing - - Biotechnological Applications, ed. A. M. Martin, pp. 273-310. Chapman and Hall, London. Lindgren, S. & Pleje, M. (1983). Silage fermentation of fish and fish waste products with lactic acid bacteria. J. Sci. Food Agric., 34, 1057-1067. Mahendrakar, N. S., Khabade, V. S., Yashoda, K. P. & Dani, N. P. (1991). Chemical and microbiological changes during autolysis of fish and poultry viscera. Trop. Sci., 31, 45-54. Mahendrakar, N. S., Rathinaraj, K., Khabade, V. S., Dani, N. P. & Ramesh, B. S. (1995). Chemical changes during fermentation of poultry intestine with molasses. Irish J. Agric. Food Res., 34, 175-181. Raa, J. & Gildberg, A. (1982). Fish silage: a review. CRC Critical Rev. in Food Sci. Nutr., 16, 383-419. Urlings, H. A. P., Bijker, P. G. J. & Van Longtestijn, J. G. (1993). Fermentation of raw poultry byproducts for animal nutrition. J. Anim. Sci., 72, 2420-2426. Zuberi, R., Fathima, R., Shamshad, S. J. & Qadri, R. B. (1993). Preparation of fish silage by microbial fermentation. Trop. Sci., 33, 171-182.