Direct processing of alginate-immobilized microalgae into polyhydroxybutyrate using marine bacterium of Saccharophagus degradans.

Alginate immobilized microalgae (AIM) was found efficient in algal cells separation and pollutants removal, however, its processing required alginate removal. In present study, polysaccharide-degrading bacterium of Saccharophagus degradans was used to biodegrade alginate and microalgae in AIM and produce polyhydroxybutyrate (PHB). Results showed that AIM cultivated in wastewater contained 34.0% carbohydrate and 45.7% protein. S. degradans effectively degraded and utilized polysaccharide of AIM to maintain five-day continuous growth at 7.1-8.8 log CFU/mL. Compared with glucose, S. degradans metabolism of mixed polysaccharide in AIM maintained the medium pH at 7.1-7.8. Increasing the inoculum concentration did not enhance AIM utilization by S. degradans due to the carbon catabolite repression of glucose which likely inactivated hydrolysis enzymes. PHB production in S. degradans peaked at 64.9 mg/L after 72 h cultivation but was later degraded to provide energy. Conclusively, S. degradans was effective in direct processing of AIM while showing potential in PHB production.

Quantitative modeling of the survival of Listeria monocytogenes in soy sauce-based acidified food products.

Primary and secondary models were developed for quantitatively characterizing the survival of Listeria monocytogenes in soy-sauce based acidified Asian style products that do not undergo a thermal treatment. The objective of this study was to quantify the effect of food matrix properties on L. monocytogenes' survival in soy sauce-based products. This quantification enables a product-specific estimation of 5-log reduction time to ensure a safe processing and management operation, to ultimately facilitate a science-based, safety-oriented product development process. A central composite design with four independent variables (pH, soy sauce, added NaCl and soluble solids) with five levels was used to plan the challenge studies on different formulations. To model microbial survival over time, different non-linear primary models were fit to the data obtained from challenge studies. The best-fit model was selected based on a series of statistical goodness-of-fit measures. Kinetic parameters estimated from the best-fit primary models were fit to response surface equations using second order polynomial regression. The best-fit primary model representative of the product formulations was a modified Weibull model. The natural logarithm of the scale parameter (δ, in h) was used as the response variable for the secondary model. This resulted in acceptable fitting compared to the observed values with R2 values of 0.95 and RMSE of 0.7 h. External validity of model predictions was conducted by comparing them to 5-log reduction times observed in independent challenge tests using different product formulations. Results indicated an acceptable validation with R2 = 0.81 and RMSE = 35 h. The present study provides quantitative tools specific for cold-fill-hold soy sauce-based products to enhance microbial safety management plans and product development.

Integration of ozone with co-immobilized microalgae-activated sludge bacterial symbiosis for efficient on-site treatment of meat processing wastewater.

Direct discharge of high concentration meat processing wastewater (MPW) into municipal sewage system will cause serious shock loading and reduce wastewater treatment efficiency, thus, efficient on-site pretreatment is usually required. Purpose of this study is to integrate ozone with microalgal biotreatment to achieve effective removal of both organic compounds and nutrients with one-step biodegradation and obtain high quality effluent dischargeable to municipal sewage system. Results showed that ozone pretreatment removed 35.0-90.2% color and inactivated 1.8-4.7 log CFU/mL bacteria in MPW. In post biotreatment using microalgae co-immobilized with activated sludge (ACS) bacteria, bacterial growth in ozone pretreated wastewater (7.1-8.1 log CFU/mL) were higher than non-pretreated control (6.0 log CFU/mL) due to enhanced biodegradability of wastewater pollutants. Algal biomass growth in wastewater pretreated with 0.5 (2489.3 mg/L) and 1 (2582.0 mg/L) minute's ozonation were improved and higher than control (2297.1 mg/L). Ozone pretreatment significantly improved nutrients removal. Following ozone pretreatment of 0.5 min, microalgal biotreatment removed 60.1% soluble chemical oxygen demand (sCOD), 79.5% total nitrogen (TN) and 91.9% total phosphate (PO43-) which were higher than control (34.4% sCOD, 63.4% TN, 77.6% total PO43-). Treated effluent contained 342.3 mg/L sCOD, 28.8 mg/L TN, 9.9 mg/L total PO43- and could be discharged into municipal sewage system. However, excessive ozone pretreatment displayed adverse impact on algal growth and sCOD removal. Therefore, integration of 0.5 min's ozone pretreatment with microalgae-based biotreatment is an efficient on-site treatment to simultaneously remove organic compounds and nutrients with one-step biodegradation.

Condensation Removal Practices and Their Potential for Contributing to Environmental Pathogen Contamination in Food Processing Facilities.

ABSTRACT: Food manufacturers often use squeegees as a tool to remove condensation from overhead surfaces. This practice is done to reduce the likelihood of environmental pathogen contamination by eliminating condensed-water droplets that could fall from overhead surfaces during production. However, this practice may actually spread environmental pathogens across these surfaces, defeating its purpose and further increasing the risk for contamination in the processing area. To understand the risk associated with this common practice, test pipes inoculated with Listeria innocua ATCC 33090 were exposed to steam to produce condensation, which was then removed by squeegees. The pipe surfaces, droplets, and squeegees were subsequently analyzed for Listeria to determine the distance the organism spread across the pipe and how many organisms were transferred to the droplets and the squeegees. Results showed that Listeria traveled as far as 16 in. across the surface of the pipe, and bacterial transfer to the droplets decreased as the squeegee traveled further from the contaminated area. Sanitizers alone were able to remove about 1 to 2 log CFU of Listeria per in2 from the squeegee blades when materials were contaminated with Listeria (>6 log CFU/in2). Among the cleaning protocols evaluated, an extensive cleaning regimen was able to remove 3 to 4 log CFU/in2, which would be recommended to reduce the risk associated with environmental pathogen transfer. This study provides evidence that supports recommendations for minimizing the cross-contamination risk associated with condensation management practices.

Reduction in pathogenic load of wheat by tempering with saline organic acid solutions at different seasonal temperatures.

As a raw agricultural commodity, wheat is exposed to microbial contamination; therefore, enteric pathogens may be among its microbiota creating a food safety risk in milled products. This research evaluates (1) the effectiveness of organic acids dissolved in saline solutions to reduce the counts of pathogenic microorganisms in soft and hard wheat, and also investigates the effect of seasonal temperature on (2) survivability of pathogens in wheat kernels and on (3) pathogen inactivation during tempering with saline organic acid solutions. Wheat samples were inoculated with cocktails of either 5 serovars of Salmonella enterica, 5 E. coli O157:H7 or 6 non-O157 Shiga toxin-producing E. coli (STEC) strains to achieve a concentration of ~7 log CFU/g. Inoculated samples were allowed to stand for 7-days at temperatures (2.0, 10.8, 24.2, 32 °C) corresponding to those experienced during winter, spring/fall, and summer (average and maximum) in the main wheat growing regions in the state of Nebraska, USA. Besides water, solutions containing acid (acetic or lactic 2.5% or 5.0% v/v) and NaCl (~26% w/v) were used for tempering the wheat to 15.0% (soft) and 15.5% (hard) moisture at the different seasonal temperatures. The survival of pathogenic microorganisms throughout the resting period, and before and after tempering was analyzed by plating samples on injury-recovery media. The survival rate of pathogenic microorganisms on wheat kernels was higher at temperatures experienced during the winter (2.0 °C) and spring/fall (10.8 °C) months. Regardless of tempering temperature, the initial pathogen load was reduced significantly by all solutions when compared to the control tempered with water (P ≤ .05). The combination of lactic acid (5.0%) and NaCl was the most effective treatment against Salmonella enterica, E. coli O157:H7 and non-O157 STEC, with average reduction values of 1.8, 1.8 and 1.6 log CFU/g for soft wheat and 2.6, 2.4 and 2.4 log CFU/g for hard wheat, respectively. Implementation of organic acids and NaCl in tempering water may have the potential to reduce the risk of pathogen contamination in milled products.

Radiofrequency pasteurization process for inactivation of Salmonella spp. and Enterococcus faecium NRRL B-2354 on ground black pepper.

Salmonella persistence in ground black pepper has caused several foodborne outbreaks and created public concern about the safety of low water activity (aw) foods. In this study, radiofrequency (RF) processing was evaluated for pasteurization of ground black pepper. Stability and homogeneity tests were done for both Salmonella spp. and E. faecium during moisture equilibration before RF heating to evaluate the inoculation method. Moisture content of samples were conditioned such that the final moisture content after RF heating reached the optimal storage moisture. RF heating was shown to provide more than 5.98 log CFU/g reduction for Salmonella spp. and the reduction of 3.89 log CFU/g for E. faecium with a 130 s of treatment time. The higher thermal resistance of E. faecium indicated its suitability as surrogate for Salmonella spp. during RF heating of ground black pepper. Piperine, total phenolics, volatile compounds, and antioxidant activity were assessed as quality parameters for ground black pepper. The results demonstrated that the RF processing provided effective inactivation of Salmonella spp. with insignificant (p > 0.05) quality deterioration.

Radio-Frequency Processing for Inactivation of Salmonella enterica and Enterococcus faecium NRRL B-2354 in Black Peppercorn.

Several Salmonella outbreaks linked to black pepper call for effective inactivation processes, because current decontamination methods result in quality deterioration. Radio-frequency (RF) heating provides a rapid heating rate and volumetric heating, resulting in a shorter come-up time. This allows for choosing a high-temperature and short-time combination to achieve the desired inactivation with minimal quality deterioration. The objectives of this study were to evaluate RF heating for inactivation of Salmonella enterica and Enterococcus faecium in black peppercorn and evaluate quality changes of RF-treated black peppercorn. Black peppercorns were inoculated with a five-strain cocktail of Salmonella or E. faecium to attain initial population levels of 6.8 and 7.3 log CFU/g, respectively, and were then adjusted to a moisture content of 12.7% (wet basis) and a water activity of 0.60 at room temperature. A stability test was performed to quantify the microbial reduction during inoculation and equilibration before RF heating inactivation. During RF heating, the cold spot was determined to be at the center on the top surface of the treated sample. In addition to inoculating the entire sample, an inoculated packed sample was placed at the cold spot of the tray. An RF heating time of 2.5 min provided a 5.31- and 5.26-log CFU/g reduction in the entire sample contained in the tray for Salmonella and E. faecium, respectively. Color parameters (L*, a*, b*), piperine content, total phenolics, scavenging activity, and most of the volatile compounds of 2.5-min RF-treated samples were not significantly different from those of the control samples. These data suggest that RF heating is a promising thermal inactivation treatment for Salmonella without significant quality deterioration, and E. faecium seems to be a suitable surrogate for Salmonella to validate the efficacy of RF heating of black peppercorn.

Response Surface Methodology for Salmonella Inactivation during Extrusion Processing of Oat Flour.

An increase in the number of foodborne outbreaks and recalls due to Salmonella in low-moisture foods has resulted in the need for the development and validation of process controls to ensure their microbiological safety. Furthermore, the Food Safety Modernization Act Preventive Controls for Human Food final rule requires food processors to validate their process controls to ensure food safety. The objective of this study was to develop a response surface model to predict Salmonella inactivation in oat flour, as affected by moisture, fat content, screw speed, and temperature. Oat flour was adjusted to different moisture (14 to 26% wet basis) and fat (5 to 15% [w/w]) contents and was then inoculated with a five-strain cocktail of Salmonella. Inoculated material was extruded through a single-screw extruder running at different screw speeds (75 to 225 rpm) and temperatures (65 to 85°C), without a die. Once steady-state conditions were attained, extruded samples were collected, cooled, and stored under refrigeration, and Salmonella survivors were enumerated. A split-plot central composite second-order response surface design was used, with the central point replicated six times. Temperature showed a significant ( P < 0.0005) positive effect on microbial reduction. Moisture content showed significant linear ( P = 0.0014) and quadratic ( P = 0.0005) effects, whereas higher fat content showed a significant ( P < 0.0001) protective effect on Salmonella destruction. The screw speed did not play a major role in inactivating Salmonella, but it had a significant ( P = 0.0004) interactive effect with temperature. Results indicated that a >5.5-log reduction was achieved in oat flour extruded at a temperature above 85°C at all moisture and fat contents evaluated at a screw speed of 150 rpm. The developed response surface model can be used to identify the extrusion process conditions to achieve a desired reduction of Salmonella based on the moisture and fat contents of the product.

A risk assessment of dietary Ochratoxin a in the United States.

Ochratoxin A (OTA) is a mycotoxin (fungal toxin) found in multiple foodstuffs. Because OTA has been shown to cause kidney disease in multiple animal models, several governmental bodies around the world have set maximum allowable levels of OTA in different foods and beverages. In this study, we conducted the first exposure and risk assessment study of OTA for the United States' population. A variety of commodities from grocery stores across the US were sampled for OTA over a 2-year period. OTA exposure was calculated from the OTA concentrations in foodstuffs and consumption data for different age ranges. We calculated the margin of safety (MOS) for individual age groups across all commodities of interest. Most food and beverage samples were found to have non-detectable OTA; however, some samples of dried fruits, breakfast cereals, infant cereals, and cocoa had detectable OTA. The lifetime MOS in the US population within the upper 95% of consumers of all possible commodities was >1, indicating negligible risk. In the US, OTA exposure is highest in infants and young children who consume large amounts of oat-based cereals. Even without OTA standards in the US, exposures would not be associated with significant risk of adverse effects.

Isolation and genetic identification of spore-forming bacteria associated with concentrated-milk processing in Nebraska.

Spore-forming bacteria are heat-resistant microorganisms capable of surviving and germinating in milk after pasteurization. They have been reported to affect the quality of dairy products by the production of enzymes (lipolytic and proteolytic) under low-temperature conditions in fluid milk, and have become a limiting factor for milk powder in reaching some selective markets. The objective of this research was to isolate and identify the population of spore-forming bacteria (psychrotrophic and thermophilic strains) associated with concentrated milk processing in Nebraska. During 2 seasons, in-process milk samples from a commercial plant (raw, pasteurized, and concentrated) were collected and heat-treated (80°C/12 min) to recover only spore-formers. Samples were spread-plated using standard methods agar and incubated at 32°C to enumerate mesophilic spore counts. Heat-treated samples were also stored at 7°C and 55°C to recover spore-formers that had the ability to grow under those temperature conditions. Isolates obtained from incubation or storage conditions were identified using molecular techniques (16S or rpoB sequencing). Based on the identification of the isolates and their relatedness, strains found in raw, pasteurized, and concentrated milk were determined to be similar. Paenibacillus spp. were associated with both raw and concentrated milk. Due to their known ability to cause spoilage under refrigeration, this shows the potential risk associated with the transferring of these problematic organisms into other dairy products. Other Bacillus species found in concentrated milk included Bacillus clausii, Bacillus subtilis, Lysinibacillus sp., Bacillus safensis, Bacillus licheniformis, Bacillus sonorensis, and Brevibacillus sp., with the last 3 organisms being capable of growing at thermophilic temperatures. These strains can also be translocated to other dairy products, such as milk powder, representing a quality problem. The results of this research highlight the importance of understanding spore-formers associated with the processing of condensed milk, which then may allow for specific interventions to be applied to control these microorganisms in this processing chain. To our knowledge, this is the first study evaluating spore-formers associated with concentrated milk in the United States.

Microbial Load of Hard Red Winter Wheat Produced at Three Growing Environments across Nebraska, USA.

Post-flowering weather variables in farm fields may influence the microbial loads of wheat grain. In this study, the effects of weather variables following wheat flowering on the microbiological quality of wheat were evaluated over two consecutive growing seasons (2011 to 2012 and 2012 to 2013) in the state of Nebraska, USA. Three hard red winter wheat lines, including two commercial cultivars (Overland and McGill) and one experimental line (NW07505), were planted in three regions with contrasting key weather variables (Southeast, South Central, and Panhandle district) to ensure that developing seeds were exposed to different weather conditions. The natural microbial flora and deoxynivalenol concentrations of 54 freshly harvested wheat samples (three samples per wheat line, with a total of 9 samples per district) were analyzed to evaluate the impacts of the weather conditions prevailing from flowering to harvesting in each growing location (district) and season on the microbiological quality and safety of wheat grain. In 2012, the values for aerobic plate counts, Enterobacteriaceae, yeasts, molds, and internal mold infection levels were significantly lower in grain samples collected from the Panhandle district than in grain harvested from the South Central and Southeastern districts. No significant differences in the yeast counts were found in grain collected from all districts in 2013, but the levels of internal mold infection and mold counts were significantly higher in grain from the Southeastern district than in grain from the Panhandle district. Deoxynivalenol was detected in all districts; however, the concentrations were below the advisory level of 1 mg/kg for processed wheat. Microbial growth during grain development seems to be dependent on the existence of a threshold level of weather variables during the season. In general, the microbial loads in wheat grain tended to be lower in those areas with lower relative humidity levels (below 55%) and with temperatures lower than 13.7°C and higher than 31.5°C.

Transmission of Escherichia coli O157:H7 to internal tissues and its survival on flowering heads of wheat.

Escherichia coli O157:H7 is a human pathogen that can cause bloody diarrhea, hemorrhagic colitis, and hemolytic uremic syndrome. E. coli O157:H7 illnesses are mainly associated with undercooked beef; however, in recent years, outbreaks have been linked to fresh produce, such as spinach, lettuce, and sprouts. In 2009, flour was implicated as the contamination source in an outbreak involving consumption of raw cookie dough that resulted in 77 illnesses. The objectives of this research were to determine (i) whether E. coli O157:H7 could be translocated into the internal tissues of wheat (Triticum aestivum) seedlings from contaminated seed, soil, or irrigation water and (ii) whether the bacterium could survive on flowering wheat heads. The levels of contamination of kanamycin-resistant E. coli O157:H7 strains in seed, soil, and irrigation water were 6.88 log CFU/g, 6.60 log CFU/g, and 6.76 log CFU/ml, respectively. One hundred plants per treatment were sown in pot trays with 50 g of autoclaved soil or purposely contaminated soil, watered every day with 5 ml of water, and harvested 9 days postinoculation. In a fourth experiment, flowering wheat heads were spray inoculated with water containing 4.19 log CFU/ml E. coli O157:H7 and analyzed for survival after 15 days, near the harvest period. To detect low levels of internalization, enrichment procedures were performed and Biotecon real-time PCR detection assays were used to determine the presence of E. coli O157:H7 in the wheat, using a Roche Applied Science LightCycler 2.0 instrument. The results showed that internalization was possible using contaminated seed, soil, and irrigation water in wheat seedlings, with internalization rates of 2, 5, and 10%, respectively. Even though the rates were low, to our knowledge this is the first study to demonstrate the ability of this strain to reach the phylloplane in wheat. In the head contamination experiment, all samples tested positive, showing the ability of E. coli O157:H7 to survive on the wheat head.

Use of Enterococcus faecium as a surrogate for Salmonella enterica during extrusion of a balanced carbohydrate-protein meal.

Multiple outbreaks of salmonellosis have been associated with the consumption of low-moisture products, including extruded products. Therefore, there is a need for a nonpathogenic, surrogate microorganism that can be used to validate extrusion processes for Salmonella. The objective of this research was to determine if Enterococcus faecium NRRL B-2354 is an adequate surrogate organism for Salmonella during extrusion. Extrusions at different temperatures were done in material contaminated with both organisms. Results indicated that the minimum temperature needed to achieve a 5-log reduction of E. faecium was 73.7°C. Above 80.3°C, the enumeration of E. faecium showed counts below the detectable levels (<10 CFU g(- 1)). Salmonella was reduced by 5 log at 60.6°C, and above 68.0°C the levels of this organism in the product were below the detection limit of the method. The data show that E. faecium is inactivated at higher temperatures than Salmonella, indicating that its use as a surrogate would provide an appropriate margin of error in extrusion processes designed to eliminate this pathogen. Attempting to minimize risk, the industry could validate different formulations, in combination with thermal treatments, using E. faecium as a safer alternative for those validation studies.

Validation of extrusion as a killing step for Enterococcus faecium in a balanced carbohydrate-protein meal by using a response surface design.

Outbreaks of salmonellosis and recalls of low-moisture foods including extruded products highlight the need for the food and feed industries to validate their extrusion processes to ensure the destruction of pathogenic microorganisms. Response surface methodology was employed to study the effect of moisture and temperature on inactivation by extrusion of Enterococcus faecium NRRL B-2354 in a carbohydrate-protein mix. A balanced carbohydrate-protein mix was formulated to different combinations of moisture contents, ranging from 24.9 to 31.1%, and each was inoculated with a pure culture of E. faecium to a final level of 5 log CFU/g. Each mix of various moistures was then extruded in a pilot scale extruder at different temperatures (ranging from 67.5 to 85°C). After the extruder was allowed to equilibrate for 10 min, samples were collected in sterile bags, cooled in dry ice, and stored at 4°C prior to analysis. E. faecium was enumerated with tryptic soy agar and membrane Enterococcus media, followed by incubation at 35°C for 48 h. Each extrusion was repeated twice, with the central point of the design being repeated four times. From each extrusion, three subsamples were collected for microbial counts and moisture determination. Based on the results, the response surface model was y = 185.04 - 3.11X(1) - 4.23X(2) + 0.02X(1)(2) - 0.004X(1)X(2) + 0.08X(2)(2), with a good fit (R(2) = 0.92), which demonstrated the effects of moisture and temperature on the inactivation of E. faecium during extrusion. According to the response surface analysis, the greatest reduction of E. faecium for the inoculation levels studied here (about 5 log) in a carbohydrate-protein meal would occur at the temperature of 81.1°C and moisture content of 28.1%. Other temperature and moisture combinations needed to achieve specific log reductions were plotted in a three-dimensional response surface graph.

Histamine and Histamine-Producing Bacteria in Retail Swiss and Low-Salt Cheeses.

Seventeen low-sodium and low-salt cheeses and 50 Swiss cheeses were surveyed for histamine and histamine-producing organisms. Two of the low-salt cheeses and nine of the Swiss cheese samples contained greater than 45 mg histamine per 100 g of cheese, as determined by the AOAC method. Over 800 total colonies were randomly chosen and screened for histamine production by the leucocrystal violet detection method following their initial isolation from MRS media. However, none of the leucocrystal violet-positive isolates from the low-salt cheese and only five from the Swiss cheese were found to produce histamine in MRS broth supplemented with L-histidine. Proteolysis (determined by the trinitrobenzene-sulfonic acid assay) was also measured in the low-salt cheeses in an attempt to further understand the role of free histidine as a substrate with respect to histamine content. In general, the cheese samples with high histamine levels also had the high values for trichloroacteic acid-soluble nitrogen. However, the highest proteolysis values did not necessarily correlate with the highest histamine values. Two samples of low-salt Swiss cheese that had high trichloroacetic acid-soluble nitrogen (greater than 200 µmoles glycine equivalents per g of cheese) contained less than 15 mg histamine per 100 g cheese.

Biogenic Amines in Cheese and other Fermented Foods: A Review.

The biogenic amine content of various foods has been widely studied because of their potential toxicity. Biogenic amines, such as tyramine and ß-phenylethylamine, have been proposed as the initiators of hypertensive crisis in certain patients and of dietary-induced migraine. Another amine, histamine, has been implicated as the causative agent in several outbreaks of food poisoning. Histamine poisoning is a foodborne chemical intoxication resulting from the ingestion of foods containing excessive amounts of histamine. Although commonly associated with the consumption of scombroid-type fish, other foods such as cheese have also been associated with outbreaks of histamine poisoning. Fermented foods such as wine, dry sausage, sauerkraut, miso, and soy sauce can also contain histamine along with other biogenic amines. Microorganisms possessing the enzyme histidine decarboxylase, which converts histidine to histamine, are responsible for the formation of histamine in foods. One organism, Lactobacillus buchneri , may be important to the dairy industry due to its involvement in cheese-related outbreaks of histamine-poisoning. The toxicity of histamine appears to be enhanced by the presence of other biogenic amines found in foods that can inhibit histamine-metabolizing enzymes in the small intestine. Estimating the frequency of histamine poisoning is difficult because most countries do not regulate histamine levels in foods, nor do they require notification when an incident of histamine poisoning occurs. Also, because histamine poisoning closely resembles a food allergy, it may often be misdiagnosed. This review will focus on the importance of histamine and biogenic amines in cheese and other fermented foods.

Histamine Production in Low-Salt Cheddar Cheese.

To assess the potential for histamine production in low-salt Cheddar cheese, pasteurized milk was inoculated with Lactobacillus buchneri St2A at levels of 102, 103, and 104 microorganisms per ml of milk. One additional vat was uninoculated and served as a control. Milk was then manufactured into low-salt (0.40%) Cheddar cheese. After 180 d of aging at 7°C, levels of L. buchneri St2A had increased approximately 100-fold in the inoculated cheese. Proteolysis, expressed as µmoles free glycine per g cheese, increased from 40 to 150 (trichloroacetic acid soluble) and from 25 to 130 (phosphotungstic acid soluble) during the ripening period. Histamine levels, however, remained low in the inoculated cheeses (<5 mg/100 g), suggesting that the potential for histamine formation may be minimal in low-salt Cheddar cheese. It was concluded that the relatively low levels of proteolysis and low temperature of storage were primarily responsible for inhibiting histamine production.