Influence of Antimicrobial Agents on the Spoilage of a Meat-Based Entomophage Diet

BIOLOGICAL AND MICROBIAL CONTROL Inﬂuence of Antimicrobial Agents on the Spoilage of a Meat-Based Entomophage Diet 1 2,3 G. D. INGLIS AND A. C. COHEN J. Econ. Entomol. 97(2): 235Ð250 (2004) ABSTRACT The microbial decomposition of a meat-based entomophage diet presented in ParaÞlm packets was investigated. Considerable bacteria but not fungi were associated with components used to prepare the diet (i.e., hensÕ eggs, liver, and ground beef). At the initial sampling time, there were no differences among diet treatments in the size of bacterial or fungal populations. Bacterial populations in diets not containing antibacterial agents rapidly increased and reached an asymptote by 24h (Ϸ1010 colony-forming units per gram). Bacterial populations also increased in diets containing antibacterial agents, but they were signiÞcantly smaller than in diets not containing antibacterial agents. The most prevalent bacteria isolated were Carnobacterium piscicola, Carnobacterium divergens, Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides, and Enterococcus spp., regardless of the antibacterial treatment used. The proliferation of fungi was delayed relative to bacteria, but signiÞcant differences were observed among the diet treatments. Fungi were most inhibited by sorbic acid and propionic acid in the absence of antibacterial agents. The most common fungi isolated were the yeasts Candida zeylanoides, Torulaspora globosa, and Yarrowia lipolytica. The pH of diets not containing antibacterial agents decreased rapidly and was highly correlated with increases in bacteria but not fungi. The results of this study demonstrate that antimicrobial agents signiÞcantly inhibit spoilage microorganisms in a meat-based diet and that alternative management strategies to delay the decomposition of such diets presented in ParaÞlm packets should target lactic acid spoilage bacteria, particularly Carnobacterium and Lactobacillus species. KEY WORDS artiÞcial diet, entomophagous, lacewings, Chrysoperla, spoilage THE PRODUCTION OF ENTOMOPHAGOUS insect species for hen 1995). The presentation of solids allows the pred- biological control is a rapidly expanding industry, and ators to recapture their digestive enzymes for further success of this industry is dependent on the econom- use in their gut, a very important aspect of their normal ical production of high-quality insects. Chrysoperla feeding process (Cohen 1998a). Based on the physi- species (lacewings) are the most widely used preda- ology of food utilization in entomophagous insects, a tory insects in biological control programs, and until semisolid diet comprised of cooked eggs, and raw liver, recently, commercial production of Chrysoperla and ground beef, brewerÕs yeast, sucrose, honey, and ace- other entomophagous insects relied primarily on the tic acid was developed by Cohen and Smith (1998). use of natural hosts. Considerable effort has gone into This diet has demonstrated its effectiveness for rearing the development of artiÞcial diets in an attempt to entomophagous insects, and it is currently being used reduce the cost of production and thereby enhance commercially to produce lacewings (Chrysoperla car- the potential for success of augmentative biological nea [Stevens] s. lat.). control programs with Chrysoperla. Despite the obvi- Despite the tremendous advantages of the meat- ous advantages of an artiÞcial diet to the industry, based diet for producing entomophagous insects, the early attempts to develop diets were met with limited requirement for ground beef, beef liver, and whole success, and as recently as 1994, no artiÞcial diet-based eggs, and other ingredients such as brewersÕ yeast, is commercial rearing of entomophages existed (Gre- not only an inviting substrate for microbial catabolism nier et al. 1994). The realization that most entomopha- but also contains many microbial contaminants (Jay gous insects, including chrysopids, use extra-oral di- 1996). Microbial contaminants have been shown to gestion to process complex solid substrates has directly and/or indirectly affect insect health. For facilitated the development of solid-based diets (Co- example, they have been shown to incite mortality (Sikorowski et al. 1980, 1992), reduce the size of in- 1 Agriculture and Agri-Food Canada, Lethbridge, AB T1J 4B1, Can- sects and delay development (Sikorowski and Thomp- ada. son 1984), cause damage to the midgut (McLaughlin 2 Biological Control and Mass Rearing Research Unit, USDAÐARS, Mississippi State, MS 39762. and Sikorowski 1978, MacGown and Sikorowski 1980), 3 Current address: Insect Diet and Rearing Institute, Tucson, AZ decrease pheromone production (Gueldner et al. 85728. 1977, Wiygul and Sikorowski 1986), decrease quanti- 236 JOURNAL OF ECONOMIC ENTOMOLOGY Vol. 97, no. 2 ties of amino acids and fatty acids within the body with heat, 1% sodium hypochlorite and/or 70% etha- (Thompson et al. 1977, Thompson and Sikorowski nol before use. The meat-based entomophage diet 1978), increase amounts of uric acid in insect blood consisted of the following ingredients: 300 g of ground (Thompson and Sikorowski 1982, 1984), and increase beef (Ϸ30% fat), 300 g of beef liver, 300 g of hensÕ eggs, oxygen utilization (Wiygul and Sikorowski 1981). Al- 45 g of sucrose, 30 g of brewerÕs yeast (Saccharomyces though the adverse effects of microbial contaminants cerevisiae), 75 g of honey (20% solution), 15 ml of on insects may be dire, the economics of diet prepa- acetic acid (10% solution), and 195 ml of tap water. ration also is an important consideration. The cost of The antimicrobial agents incorporated into the diet the diet ingredients and of the labor involved with its were the antibacterial agents streptomycin sulfate (0.3 preparation are signiÞcant, and increasing the time g) (Sigma-Genosys, St. Louis, MO) and chlortetracy- that insects can feed on the diet without compromis- cline (0.3 g) (Sigma-Genosys) and the antifungal ing their health (i.e., due to microbial spoilage) will agents propionic acid (1.8 g) (Sigma-Genosys) and have a positive impact on the economics of produc- potassium sorbate (1.8 g) (Sigma-Genosys). Initially, tion. For example, packets containing the meat-based the ground beef, beef liver, brewerÕs yeast, honey entomophage diet are placed on the top of cages solution, and 60 ml of tap water were homogenized in containing lacewings, and the packets are replaced a Hamilton Beach blender (high setting) for Ϸ2 min. approximately every 48 h due to compromised quality. Representative samples of ground beef, liver, eggs Delaying the replacement time by even 24h would (i.e., obtained from a local grocery store), and tap substantially decrease costs of production. water (containing honey) were taken before their Antimicrobial agents, including the antibiotic placement in the blender. In a 4-liter beaker, 135 ml of agents streptomycin sulfate and chlortetracycline, are water was heated to 85Ð95ЊC, and sucrose was added currently incorporated into the diet in an attempt to and allowed to dissolve. Acetic acid and the appro- suppress spoilage microorganisms. These antibiotics priate antimicrobial agents (i.e., according to treat- have a minimal impact on C . carnea larvae (Cohen and ment) were subsequently added to the sucrose solu- Smith 1998), but their efÞcacy in preventing spoilage tion, followed by the hensÕ eggs. In a preliminary has not been investigated. In the case of the meat- experiment, the sucrose and acetic acid solutions were based entomophage diet, conventional sterilization shown to be free of microorganisms. Given the small and pasteurization methods cannot be used because amount of antimicrobial agents added (Ͻ0.2%), no the chemical and physical changes that occur are very attempt was made to compensate for volume differ- detrimental to Chrysoperla larvae (A.C.C., unpub- ences in diet treatments missing two or more agents. lished data). In the majority of diets, the microbiology The mixture was stirred constantly until the eggs had of spoilage is poorly understood, and insect diet pro- gelled but were of a sticky, stringy consistency (Ϸ2Ð3 fessionals often rely on a relatively limited number of min). The meat was then added to the hot egg slurry wide-spectrum antimicrobial agents. To illustrate this, and blended for 3Ð5 min until the entire mixture was in a survey of 58 synthetic diets used for phytophagous of a stringy, paste-like consistency. The following an- insects, 32% used chlortetracycline, 100% used meth- timicrobial treatments were included in the experi- yl-p-hydroxybenzoate, 10% used sodium benzoate and ment: 1) diet one contained both antibacterial (strep- formaldehyde, and 72% used sorbic acid (Singh and tomycin sulfate and chlortetracycline) and antifungal House 1970). A reliance on antimicrobial agents is not agents (propionic acid and potassium sorbate); 2) diet without its problems. Antibiotics are expensive, and two contained antibacterial agents only; 3) diet three they can be toxic to insects (Sikorowski et al. 1984). contained antifungal agents only; and 4) diet four Furthermore, serious concerns exist about the poten- contained neither antibacterial or antifungal agents. tial development of bacterial resistance, particularly The order in which the diet treatments were prepared with regard to the prophylactic use of antibiotics. was randomly selected in each replicate, and prepared Research to elucidate the microbial taxa responsible diets were stored aseptically at 5ЊC until they could be for spoilage is a prerequisite for the

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