Jayne Stratton

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 β-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.

High Cell-Density Fermentation

The purpose of this chapter is to educate the reader about the basic equipment and strategies used in fermentations of P. pastoris in both bench-top and pilot-scale operations. A key element in expression of foreign proteins in this yeast is the need for sufficient aeration, which is achieved by proper mixing of the media and by blending gases to control dissolved oxygen content. Automatic pH control is essential for growth and expression in P. pastoris. Finally, fed-batch fermentations require the use of peristaltic pumps and tubing capable of low rates of delivery for the feeding of nutrients and base. Teflon tubing and peristaltic pump adapters are recommended for fed-batch operations. The information in this chapter should enable a reader with little or no experience to perform a high-cell density fermentation of a P. pastoris expression strain. Although most procedures described here are specifically for the BioFlo III (NBS), it should be possible to achieve high expression levels with almost any good-quality fermentor, modified to accommodate this organism.

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.

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.

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 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.

Reduction in Microbial Load of Wheat by Tempering with Organic Acid and Saline Solutions

Reducing microbial contamination in wheat is desirable to ensure consumer safety. In this study, the efficacy of adding organic acids and NaCl to tempering water to reduce microbial contamination in hard wheat was evaluated. Hard red winter wheat was tempered to 15.5% moisture by adding sterile distilled water (control) or tempering solutions containing organic acids (acetic, citric, lactic, or propionic; 1.0, 2.5, or 5.0 v/v), NaCl (26 or 52% w/v), or a combination of organic acid (acetic or lactic; 2.5 and 5.0% v/v) and NaCl (26 or 52% w/v). After tempering, the microbial load was significantly reduced by all the acid and NaCl treatments when compared with the control. Wheat tempered with 5% acetic, propionic, and lactic acids resulted in reductions of 1.7, 2.3, and 3.8 log colony forming units (CFU) per gram in aerobic plate count (APC), Enterobacteriaceae (Eb), and mold counts, respectively. The combination lactic acid (5.0%) and NaCl (52%) was the most effective against APC and Eb, with an average re...

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 Essential Oils and Plant Extracts to Control Microbial Contamination in Pet Food Products

The antimicrobial activity of 25 essential oils and plant extracts against Salmonella Typhimurium and Penicillium roqueforti was tested by disc diffusion and quantified by agar dilution. Afterwards, the effect of the most promising essential oils was studied in an extruded pet food product over 21 days, with the oil mixed either into the product or as part of its coating. In vitro, the best inhibitors for Salmonella were cinnamon essential oil (EO) at 0.05% and thyme EO at 0.1%, while P. roqueforti was best inhibited by cinnamon EO at 0.01% and spearmint EO at 0.5%. When tested in the extruded product, cinnamon EO (0.05% and 0.1%) and spearmint EO (0.5%) proved ineffective against P. roqueforti, either mixed into the product or as part of its coating. Only cinnamon EO (0.1%) as part of the coating was able to reduce Salmonella significantly faster than the control (p-value=0.0408) during storage for 21 days. Based on the results, spice essential oils can act as inhibitors of Salmonella in a pet food product, when present at a sufficient concentration.

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.