Antifungal activities of combined treatments of irradiation and essential oils (EOs) encapsulated chitosan nanocomposite films in in vitro and in situ conditions.

Cellulose nanocrystals (CNCs) reinforced chitosan based antifungal films were prepared by encapsulating essential oils (EOs) nanoemulsion. Vapor phase assays of the chitosan-based nanocomposite films loaded with thyme-oregano, thyme-tea tree and thyme-peppermint EO mixtures showed significant antifungal activity against Aspergillus niger, Aspergillus flavus, Aspergillus parasiticus, and Penicillium chrysogenum, reducing their growth by 51-77%. Combining the bioactive chitosan films loaded with thyme and oregano EOs produced ~2 log reduction in fungal growth in inoculated rice during 8 weeks of storage at 28 °C. The bioactive films showed a slow release (26%) of volatile components over 12 weeks of storage. Sensorial evaluation of rice samples packed with the bioactive films showed no significant change in odor, taste, color and general appreciation compared with untreated rice. Incorporation of cellulose nanocrystals (CNCs) with the chitosan matrix played an important role in stabilizing the physicochemical and release properties of the nanocomposite films. In addition, combining the bioactive chitosan films with a dose of 750 Gy of ionizing radiation showed significantly higher antifungal and mechanical properties than treatment with the bioactive film or irradiation alone.

Cancer Chemopreventive, Antiproliferative, and Superoxide Anion Scavenging Properties of Kluyveromyces marxianus and Saccharomyces cerevisiae var. boulardii Cell Wall Components.

This study investigated the cancer chemopreventive, the antiradical, and the antiproliferative properties of polysaccharides extracts from cell wall of Saccharomyces boulardii and Kluyveromyces marxianus. ß-glucan, mannan, and chitin were also quantified to identify the most important extract responsible for these biological properties. Soluble and insoluble glucans as well as mannoprotein were extracted from cell wall using single hot-alkaline method. Superoxide anion scavenging (antiradical capacity), NAD(P)H: quinone reductase (QR) (EC 1.6.99.2) induction, and antiproliferative assays were done for the evaluation of biological properties of those extracts. The insoluble glucan from S. boulardii revealed the most relevant biological properties by increasing QR activity and exhibiting the highest growth inhibition against colorectal cancer cells. Moreover, high amount of glucan, high glucan/total sugars ratios, and low chitin/glucan ratios were shown to have an impact on enhancing cancer chemopreventive and antiproliferative properties. To our knowledge, this is the first study that demonstrates QR activity by yeast cell wall components in a dose-dependent manner.

Antilisterial effects of antibacterial formulations containing essential oils, nisin, nitrite and organic acid salts in a sausage model.

This study was conducted to evaluate the effects of sixteen antibacterial formulations against Listeria monocytogenes in a sausage model using a standard experimental design with 4 independent factors at 2 levels (2(4)). Four independent factors consisted of nisin (12.5-25 ppm), nitrite (100-200 ppm) and organic acid salts (1.55-3.1 %) and the mixture of Chinese cinnamon and Cinnamon bark Essential Oils (EOs) (0.025-0.05 %). Based on the analysis, utilization of low (0.025 %) or high concentration (0.05 %) of EOs in combination with low concentration of nitrite (100 ppm), organic acid salts (1.55 %), and nisin (12.5 ppm) could reduce respectively 1.5 or 2.6 log CFU/g of L. monocytogenes in sausage at day 7 of storage as compared to the control. A predictive equation was created to predict the growth of L. monocytogenes in sausage. The sensory evaluation was then performed on selected optimized formulations in cooked meat (both pork and beef sausages) with a trained jury consisting of 35 individuals, demonstrated the selected antimicrobial formulations were organoleptically acceptable. The results revealed an important role of hurdle technology to control L. monocytogenes in meat product.

Enhancement of Nisin Production by Lactococcus lactis subsp. lactis.

Lactococcus lactis subsp lactis BSA (L. lactis BSA) was isolated from a commercial fermented product (BSA Food Ingredients, Montreal, Canada) containing mixed bacteria that are used as starter for food fermentation. In order to increase the bacteriocin production by L. lactis BSA, different fermentation conditions were conducted. They included different volumetric combinations of two culture media (the Man, Rogosa and Sharpe (MRS) broth and skim milk), agitation level (0 and 100 rpm) and concentration of commercial nisin (0, 0.15, and 0.30 µg/ml) added into culture media as stimulant agent for nisin production. During fermentation, samples were collected and used for antibacterial evaluation against Lactobacillus sakei using agar diffusion assay. Results showed that medium containing 50 % MRS broth and 50 % skim milk gave better antibacterial activity as compared to other medium formulations. Agitation (100 rpm) did not improve nisin production by L. lactis BSA. Adding 0.15 µg/ml of nisin into the medium-containing 50 % MRS broth and 50 % skim milk caused the highest nisin activity of 18,820 AU/ml as compared to other medium formulations. This activity was 4 and ~3 times higher than medium containing 100 % MRS broth without added nisin (~4700 AU/ml) and 100 % MRS broth with 0.15 µg/ml of added nisin (~6650 AU/ml), respectively.

Antimicrobial effects of marine algal extracts and cyanobacterial pure compounds against five foodborne pathogens.

The marine environment is a proven source of structurally complex and biologically active compounds. In this study, the antimicrobial effects of a small collection of marine-derived extracts and isolates, were evaluated against 5 foodborne pathogens using a broth dilution assay. Results demonstrated that algal extracts from Padina and Ulva species and cyanobacterial compounds antillatoxin B, laxaphycins A, B and B3, isomalyngamide A, and malyngamides C, I and J showed antimicrobial activity against Gram positive foodborne pathogens (Listeria monocytogenes, Bacillus cereus and Staphylococcus aureus) at low concentrations (⩽ 500 µg/ml). None of the algal extracts or cyanobacterial isolates had antibacterial activity against Gram negative bacteria (Escherichia coli and Salmonella enterica serovar Typhimurium).

Development of a model describing the inhibitory effect of selected preservatives on the growth of Listeria monocytogenes in a meat model system.

The objective of this study was to evaluate the impact of seven independent factors consisting of sodium nitrite, pH, sodium chloride, sodium acetate, sodium lactate syrup, calcium propionate and a blend of nisin and hop alpha acids on the growth rate of Listeria monocytogenes in ham as a model of ready-to-eat (RTE) meat products. A central composite consisted of seven factors mentioned above was designed and the response surface methodology was applied for creating a mathematic model to predict the growth rate of L. monocytogenes in RTE meat products. Six parameters showed a significant (P ≤ 0.1) influence on the growth rate of L. monocytogenes. Only the blend of nisin and hop alpha acids did not show any significant effect (P > 0.1) in the concentrations used in this study. Increasing concentration of sodium chloride, sodium nitrite, sodium acetate, potassium lactate and calcium propionate in meat reduced bacterial growth rate while increasing pH in meat increased the growth rate of L. monocytogenes. The current mathematical equation will be an important tool in order to reduce the required number of challenge studies performed in order to ensure a safe food product.

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of Lactococcus lactis MM19 and Pediococcus acidilactici MM33.

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe--MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract-TGE broth).

Antimicrobial Effects of Nisin, Essential Oil, and γ-Irradiation Treatments against High Load of Salmonella typhimurium on Mini-carrots.

UNLABELLED: This study aimed at using essential oil (EO) alone or combined EO with nisin and γ-irradiation to control Salmonella Typhimurium during the refrigerated storage of mini-carrots. Peeled mini-carrots were inoculated with S. Typhimurium at a final concentration of approximately 7 log CFU/g. Inoculated samples were coated by 5 different coating solutions: (i) nisin solution at final concentration of 10(3) IU/mL; (ii) mountain savory EO solution at 0.35%; (iii) carvacrol solution at 0.35%; (iv) mountain savory EO at 0.35% plus nisin solution of 10(3) IU/mL; or (v) carvacrol at 0.35% plus nisin solution of 10(3) IU/mL. Coated mini-carrots were then irradiated at 0.5 or 1.0 kGy and compared to an unirradiated control sample. Samples were kept at 4 °C and microbial analyses were conducted at days 1, 3, 6, and 9. The results showed that mini-carrots coated by carvacrol plus nisin solution or mountain savory EO plus nisin solution in combination with irradiation at 1.0 kGy completely eliminated S. Typhimurium to under the detection limit during the storage. Thus, the combined treatments using carvacrol plus nisin or mountain savory EO plus nisin coating solution and irradiation at 1.0 kGy could be used as an effective method for controlling S. Typhimurium in mini-carrots. PRACTICAL APPLICATION: This study shows the effect of using γ irradiation at low dose (1 kGy) to reduce significantly the growth of Salmonella typhimurium in mini-carrots. It also shows that combination of nisin, essential oils with γ irradiation have the best antibacterial effects against S. Typhimurium during the storage of mini-carrots. The results can be used for practical application in food industry in terms of food safety.

Antimicrobial effects of essential oils, nisin, and irradiation treatments against Listeria monocytogenes on ready-to-eat carrots.

The study aimed at using essential oil (EO) alone or combined EO with nisin and low dose γ-irradiation to evaluate their antibacterial effect against Listeria monocytogenes during storage of carrots at 4 °C. Minicarrots were inoculated with L. monocytogenes at a final concentration of approximately 7 log CFU/g. Inoculated samples were coated by nisin at final concentration of 10(3) International Unit (IU)/mL or individual mountain savory EO or carvacrol at final concentration of 0.35%, w/w) or nisin plus EO. The samples were then irradiated at 0, 0.5, and 1.0 kGy. The treated samples were kept at 4 °C and microbial analysis of samples were conducted at days 1, 3, 6, and 9. The results showed that coating carrots by carvacrol plus nisin or mountain savory plus nisin and then irradiating coated carrots at 1 kGy could reduce L. monocytogenes by more than 3 log at day 1 and reduced it to undetectable level from day 6. Thus, the combined treatments using nisin plus carvacrol or nisin plus mountain savory and irradiation at 1.0 kGy could be used as an effective method for controlling L. monocytogenes in minicarrots.

Synergistic effect of gamma (γ)-irradiation and microencapsulated antimicrobials against Listeria monocytogenes on ready-to-eat (RTE) meat.

Oregano essential oil (Origanum compactum; 250 µg/ml), cinnamon essential oil (Cinnamomum cassia; 250 µg/ml) and nisin (16 µg/ml) were used alone or in combination to evaluate their efficiency to inhibit the growth of Listeria monocytogenes on RTE ham. Microencapsulation of the antimicrobial formulations was done to verify the potential effect of the polymer to protect the antimicrobial efficiency during storage. Combined treatments of antimicrobial formulation with γ-irradiation were done to verify the synergistic effect against L. monocytogenes. Microencapsulation of essential oils-nisin and γ-irradiation treatment in combination showed synergistic antimicrobial effect during storage on RTE meat products. Microencapsulated cinnamon and nisin in combination with γ-irradiation (at 1.5 kGy) showed 0.03 ln CFU/g/day growth rate of L. monocytogenes whereas the growth rate of non-microencapsulated cinnamon and nisin in combination with γ-irradiation was 0.17 ln CFU/g/day. Microencapsulation significantly (P ≤ 0.05) improved the radiosensitivity of L. monocytogenes. Microencapsulated oregano and cinnamon essential oil in combination with nisin showed the highest bacterial radiosensitization 2.89 and 5, respectively, compared to the control.

Antibacterial and physical effects of modified chitosan based-coating containing nanoemulsion of mandarin essential oil and three non-thermal treatments against Listeria innocua in green beans.

The antimicrobial activity against Listeria innocua of three different combined non-thermal treatments, along with the impact on color and texture on green bean samples, was evaluated. In this study a bioactive coating formulation based on modified chitosan containing 0.05% nanoemulsion of mandarin essential oil was tested in combination with γ-irradiation, UV-C and ozonated water treatments, and the results in terms of antimicrobial activity, color and texture changes, were evaluated during 14 days storage. The combined coating and γ-irradiation treatment gave promising results, showing 3.3 log CFU/g initial microbial reduction, and exhibiting a strong synergistic antimicrobial effect. The treatment based on UV-C and coating formulation allowed a 3 log CFU/g reduction of initial L. innocua population on samples, showing a good residual antimicrobial activity and preventing loss of firmness and color changes during storage. The combined treatment of coating and ozonated water did not show any synergistic or additive antimicrobial effect, but they showed an impact on firmness and color. In conclusion UV-C and γ-irradiation treatments, in combination with the bioactive coating, represent an effective approach to control the growth of L. innocua on vegetable foods.

Effect of γ-irradiation on gene expression of heat shock proteins in the foodborne pathogen Escherichia coli O157:H7.

PURPOSE: The expression levels of seven genes (clpB, dnaK, groES, grpE, htpG, htpX and ibpB) encoding heat shock proteins (HSP) in Escherichia coli O157:H7 (E. coli) gamma irradiated was investigated. Timing impact of post-irradiated RNA extraction on the expression levels of these seven genes was also studied at a dose damaging the bacterial cells (0.4 kGy). METHODS: Bacterial samples were γ-irradiated at 0.4 kGy and at a lethal dose of 1.3 kGy. RNA was extracted at 0 min post irradiation for both irradiation doses and at 15, 30, 60, 90 or 120 min post-irradiation at the dose damaging the cells. Quantification of the gene expression was performed using quantitative real-time polymerase chain reaction (q-RT-PCR). RESULTS: The expression of genes encoding HSP was a very dynamic process evolving rapidly when E. coli cells were irradiated at 0.4 kGy. Notably, groES, grpE and ibpB were more up- regulated at 1.3 kGy than those at 0.4 kGy. CONCLUSIONS: For the seven genes studied there were more damaged proteins during irradiation at the lethal dose and this dose causes increased expression in HSP which contributes to damage reparation. Expression patterns of genes encoding HSP in E. coli treated by γ-irradiation are different from those treated by heat shock.

In vitro evaluation of antimicrobial activities of various commercial essential oils, oleoresin and pure compounds against food pathogens and application in ham.

The purpose of this research was to evaluate the application of commercially available essential oils (EOs) and oleoresins to control bacterial pathogens for ready to eat food. In this study, sixty seven commercial EOs, oleoresins (ORs) and pure compounds were used to evaluate in vitro their antimicrobial activity against six food pathogens. These products were first screened for their antimicrobial activity using disk diffusion assay. Forty one products were then chosen for further analysis to determine their minimum inhibitory concentration against 6 different bacteria. There were 5 different products (allyl isothiocyanate, cinnamon Chinese cassia, cinnamon OR, oregano and red thyme) that showed high antimicrobial activity against all tested bacteria. Further analysis examined the effect of four selected EOs on controlling the growth rate of mixed cultures of Listeria monocytogenes in ham. A reduction of the growth rate by 19 and 10% was observed when oregano and cinnamon cassia EOs were respectively added in ham at a concentration of 500 ppm.

Effect of combination of essential oils and bacteriocins on the efficacy of gamma radiation against Salmonella Typhimurium and Listeria monocytogenes.

PURPOSE: Using essential oils (EO) alone or combined EO with nisin to enhance the lethality of Salmonella Typhimurium and Listeria monocytogenes to γ-irradiation treatment. MATERIALS AND METHODS: Cell suspension or inoculated carrots (10(6) colony forming unit (CFU)/ml) of L. monocytogenes or Salmonella Typhimurium were treated with carvacrol and mountain savory EO alone or treated by the combined EO plus nisin and were irradiated at different doses. The radiation dose required to reduce bacterial population by 90% (D(10) value) and the relative sensitivity (RS) of treated bacteria to irradiation were calculated. RESULTS: The RS of L. monocytogenes cell suspension to irradiation was increased by 4.19 and 6.31 times when treated by carvacrol plus nisin and mountain savory plus nisin, respectively, as compared to the control. The RS of Salmonella Typhimurium cell suspension to irradiation was increased by 1.84-fold when treated with carvacrol plus nisin. Coated carrots by nisin plus carvacrol increased the RS of L. monocytogenes to irradiation by 2.74 times while coated carrots by nisin and nisin plus carvacrol increased the RS of Salmonella Typhimurium to irradiation by 2.46 and 2.52, respectively. CONCLUSIONS: The combined EO with nisin and irradiation could increase the RS of L. monocytogenes and Salmonella Typhimurium.

Partial Characterization of Bacteriocins Produced by Two New Enterococcus faecium Isolated from Human Intestine.

This study aimed at characterizing two novel bacteriocin-producing enterococcal strains isolated from human intestine. A total of 200 lactic acid bacteria were isolated from a woman stool sample. Two of them were selected for characterization due to their high antimicrobial activity against five strains of Listeria monocytogenes. The selected bacteria were identified as two different strains of Enterococcus faecium and designated MT 104 and MT 162. The bacteriocins produced by MT 104 and MT 162 were stable at different pH ranging from 2 to 11 and were active after different treatments such as heat, enzymes, detergents, and γ-irradiation. The two isolated strains exhibited some probiotic properties such as survival in simulated gastric fluid and intestinal fluid, lack of expression of bile salt hydrolase or hemolytic activity, adhesion to Caco-2 cells efficiently, and sensitivity to clinical antimicrobial agents. Thus, the two isolated strains of E. faecium could become new probiotic bacteria and their bacteriocins could be used for controlling L. monocytogenes in combination with irradiation for food preservation.

Response of Bacillus cereus vegetative cells after exposure to repetitive sublethal radiation processing in combination with nisin.

The present study evaluated the response of Bacillus cereus LSPQ 2872 vegetative cells, following exposure to single and repetitive sublethal γ-radiation treatment at 1 kGy alone or in combination with nisin at its maximum tolerated concentration, in BHI broth supplemented with 0.5% glucose. Results showed that B. cereus has the capability to develop increased resistance to subsequent cycles of gamma irradiation and a significant increase (p ≤ 0.05) of the relative radiation resistance (D(10)) value was noticed after the fourth irradiation at 1 kGy. The sequence and the repetitive sublethal treatment of γ-radiation with nisin affected significantly (p ≤ 0.05) B. cereus radio-tolerance since lower D(10) values were recorded. Our results showed that the response of B. cereus to repetitive mild bactericidal treatment was accompanied by relevant modifications in the cell properties leading to the increased resistance. Transmission electron microscopy revealed that resistance might be related to changes in the cell wall. Multiparameter flow cytometry revealed effectively diverse physiological changes that B. cereus underwent during development of radio-resistance. The development of increased resistance to gamma irradiation was accompanied by an increase of the percentage of injured and viable cells at the expense of dead cells. Nisin treated cells developed also increased radio-resistance if repetitively processed with γ-radiation at sublethal doses which was not demonstrated by plate counting.

Mathematical relationships between spore concentrations, delta-endotoxin levels, and entomotoxicity of Bacillus thuringiensis preparations produced in different fermentation media.

Mathematic relationships between spore concentrations, delta-endotoxin concentrations and entomotoxicity (Tx) of Bacillus thuringiensis var. kurstaki HD-1 (Btk HD-1) preparations produced in six different media were analysed. The relationship between delta-endotoxin and spore concentration and SpTx-spore (specific Tx per 1000 spore) and spore concentration produced in the different media (starch industry wastewater (SIW) with total solids (TS) concentration of 15g/L, SIW with TS of 30g/L, SIW supplemented with 0.2% (w/v) colloidal chitin, SIW supplemented with 1.25% (w/v) cornstarch and 0.2% (v/v) Tween 80, secondary sludge, and semi-synthetic medium) strictly followed the Power law. Tx and delta endotoxin concentration followed the exponential relation whereas a definite relation between Tx and spore concentration could not be established. Spore and delta-endotoxin produced at the early time (12h) during fermentation might be more toxic than those produced during latter period of fermentation irrespective of media used. Tx and delta-endotoxin concentration exhibited a semi-log linear relationship. Based on these findings, delta-endotoxin concentration can be determined rapidly to monitor the progress of the biopesticide production process.

Capillary electrophoresis separation of protein composition of γ-irradiated food pathogens Listeria monocytogenes and Staphylococcus aureus.

BACKGROUND: A capillary electrophoresis method using UV detection was developed to analyse protein composition of the lysates of two foodborne pathogens, Listeria monocytogenes and Staphylococcus aureus which were previously treated at different irradiation doses. METHODOLOGY AND PRINCIPAL FINDINGS: Bacterial samples were γ-irradiated at different doses to produce damage cells, to kill cells and to provoke viable but non culturable cells (VBNC) in order to evaluate the respective expression of stress proteins. In Listeria monocytogenes, two proteins (MW of 70.2 and 85.4 kDa) were significantly changed (P ≤ 0.05) at different doses of irradiation. In Staphyloccocus aureus, one protein (50 S ribosomal protein) with the MW of 16.3 kDa was significantly decreased at a low dose of irradiation treatment and the other protein (transcriptional regulator CtsR) with the MW of 17.7 kDa was increased significantly (P ≤ 0.05) at all doses of irradiation treatment compared to control. CONCLUSION: Expression of two proteins from the acyltransferase family in Listeria monocytogenes was statistically changed during irradiation treatment (P ≤ 0.05). In Staphylococcus aureus, expression of the 50 S ribosomal protein decreased and the transcriptional regulator CtsR espression increased significantly (P ≤ 0.05) following irradiation treatment. These expressed proteins do not belong to the well-known heat shock proteins family of Listeria monocytogenes and Staphylococcus aureus. The research further confirmed that capillary electrophoresis is a useful method to separate and analyse proteins expression which may be related to the resistance or sensitivity of food pathogens to γ-irradiation.

Batch and fed-batch fermentation of Bacillus thuringiensis using starch industry wastewater as fermentation substrate.

Bacillus thuringiensis var. kurstaki biopesticide was produced in batch and fed-batch fermentation modes using starch industry wastewater as sole substrate. Fed-batch fermentation with two intermittent feeds (at 10 and 20 h) during the fermentation of 72 h gave the maximum delta-endotoxin concentration (1,672.6 mg/L) and entomotoxicity (Tx) (18.5 x 10(6) SBU/mL) in fermented broth which were significantly higher than maximum delta-endotoxin concentration (511.0 mg/L) and Tx (15.8 x 10(6) SBU/mL) obtained in batch process. However, fed-batch fermentation with three intermittent feeds (at 10, 20 and 34 h) of the fermentation resulted in the formation of asporogenous variant (Spo-) from 36 h to the end of fermentation (72 h) which resulted in a significant decrease in spore and delta-endotoxin concentration and finally the Tx value. Tx of suspended pellets (27.4 x 10(6) SBU/mL) obtained in fed-batch fermentation with two feeds was the highest value as compared to other cases.

Induced production of chitinase to enhance entomotoxicity of Bacillus thuringiensis employing starch industry wastewater as a substrate.

Induced production of chitinase during bioconversion of starch industry wastewater (SIW) to Bacillus thuringiensis var. kurstaki HD-1 (Btk) based biopesticides was studied in shake flask as well as in computer-controlled fermentors. SIW was fortified with different concentrations (0%; 0.05%; 0.1%; 0.2%; 0.3% w/v) of colloidal chitin and its consequences were ascertained in terms of Btk growth (total cell count and viable spore count), chitinase, protease and amylase activities and entomotoxicity. At optimum concentration of 0.2% w/v colloidal chitin, the entomotoxicity of fermented broth and suspended pellet was enhanced from 12.4x10(9) (without chitin) to 14.4x10(9) SBU/L and from 18.2x10(9) (without chitin) to 25.1x10(9) SBU/L, respectively. Further, experiments were conducted for Btk growth in a computer-controlled 15 L bioreactor using SIW as a raw material with (0.2% w/v chitin, to induce chitinase) and without fortification of colloidal chitin. It was found that the total cell count, spore count, delta-endotoxin concentration (alkaline solubilised insecticidal crystal proteins), amylase and protease activities were reduced whereas the entomotoxicity and chitinase activity was increased with chitin fortification. The chitinase activity attained a maximum value at 24 h (15 mU/ml) and entomotoxicity of suspended pellet reached highest (26.7x10(9) SBU/L) at 36 h of fermentation with chitin supplementation of SIW. In control (without chitin), the highest value of entomotoxicity of suspended pellet (20.5x10(9) SBU/L) reached at 48 h of fermentation. A quantitative synergistic action of delta-endotoxin concentration, spore concentration and chitinase activity on the entomotoxicity against spruce budworm larvae was observed.