Nuts and Grains: Microbiology and Preharvest Contamination Risks

ABSTRACT

Low-water-activity foods have been involved in recalls and foodborne disease outbreaks. Increased consumption; better detection methods and reporting systems; improved surveillance, trace-back, and ability to connect sporadic foodborne illnesses; and inadequate implementation of food safety programs are some of the likely reasons for the increase in frequency of recalls and outbreaks linked to dry foods. Nuts and grains can be contaminated with foodborne pathogens at any stage during production, processing, storage, and distribution. Focusing on preharvest contamination, the various potential sources of contamination include soil, animal intrusion, contaminated harvesting equipment, harvest and preharvest handling, storage conditions, and others. The low water activity of nuts and grains prevents the growth of most foodborne pathogens on their surfaces. The long-term survival of bacterial foodborne pathogens (Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes) on dry foods has been documented in the literature for different nut types. Preventing contamination is the key to avoiding foodborne disease risks linked to dry foods. The implementation of good agricultural practices and other food safety systems provides a proactive approach to address concerns thoroughly. A plethora of research is available on preventing the growth of mycotoxin-producing fungi on the surface of nuts and grains. Milling is an effective mechanism to reduce the microbial load on grains. This review focuses on providing information about associated foodborne microorganisms, preharvest contamination sources, and good agricultural practice recommendations for nuts and grains.

INTRODUCTION

Almonds, walnuts, and cashews are the major tree nuts produced in the world. The United States is the largest producer of tree nuts (1). Peanuts, also known as groundnuts, are produced largely in China, followed by India (1). Almonds are the major nuts produced by the United States, followed by peanuts, walnuts, and pistachios in this order (1). According to the 2014–2015 Global Statistical Review and world nut and dried fruit map, the world’s tree nut production increased by 5.4% compared to the previous season (1). By definition, nuts can be divided into several categories, including drupes (e g., almonds, coconuts, pecans, pistachios, walnuts), legumes (e.g., peanuts), nuts (e.g., acorns, chestnuts, hazelnuts), and seeds (e.g., brazil nuts, cashews, pumpkin, pine nuts, sesame, sunflower) (2). Nuts are consumed as a whole or as an ingredient in confectionary, bakery, and snack products. Nuts can be consumed raw or can be processed by thermal treatments such as oil roasting, dry roasting, blanching, and others.

Cereal grains are an important food resource worldwide (3). Rice, wheat, and corn are the major grains produced around the world. Sorghum and millet are also produced in several regions, with the production being much lower than the three major crops (3). Global cereal production is expected to decline by 1.2 million tons (0.6%) in 2017 compared to the 2016 crop, with the production in 2017 predicted to be 2,593 million tons (4). Approximately 60% of the calories consumed in developing countries and 30% of calories consumed in developed countries are derived from cereals (3). Grains are consumed as a whole or in refined form. Many whole grains are a rich source of dietary fiber (5).

NUT PRODUCTION, MICROBIOLOGY, AND CONTAMINATION SOURCES

Tree nuts take years to bear fruit after planting. The production, harvesting, and postharvest practices vary among different types of tree nuts. With most of the production and harvesting conducted mechanically, the degree of mechanization can vary (6).

Almond production in the United States primarily occurs in California (1). Almond production includes irrigation of crops using furrow, sprinkler, and drip irrigation. Nitrogen, potassium, and zinc are the major nutrients applied to the crop (7). Almond harvesting is a well-mechanized process involving mechanical tree shakers and other equipment. Almonds are allowed to dry on ground for a few days to 2 weeks after knocking the nuts (8). Nuts are swept into windrows, placed onto trailers, and transported to hullers. When rainy conditions prevail, nuts should be dried prior to hulling. Drying can be done using forced hot air at 49 to 54°C in a batch dryer or at 82°C in a continuous flow dryer. After removal of the hulls, nuts are placed in bulk storage and transported to the processing plant (8).

Hazelnuts, or filberts, are native tree nuts of Europe and adjacent areas (9), and in the United States the majority of production occurs in Oregon (1). About 70% of the world production of hazelnuts occurs in Turkey (9). Hazelnuts are best suited for well-drained soils with high environmental humidity conditions (10). The U.S. production (99%) of hazelnuts is concentrated in Oregon due to suitable weather conditions (10). Oregon rainfall is the major source of water for hazelnuts due to the good water-holding capacity of the clay soil (10). Nitrogen and potassium are the main fertilizers applied to hazelnuts (10). Mechanical tree shakers are used for harvesting of hazelnuts, followed by arranging the nuts in the center of rows using a sweeper. Nuts are then transported to the nut dryer or processor.

Macadamias are native tree nuts of Australia (9). U.S. macadamia production is primarily concentrated in Hawaii (1). Water and fertilizer requirements of macadamia nuts vary based on the production regions. During harvest, macadamia nuts are allowed to fall onto the ground naturally and are picked from the ground. The flowering of macadamia nuts takes place over several months, which makes it difficult to predict the exact harvest month of nuts. Mechanical tree shakers are not very useful for macadamia nuts (6). Upon harvesting, macadamia nuts are dried using a two-stage process. The first stage reduces the moisture content of nuts to 14%, and the second stage brings the moisture content down to 4%.

Pecans are the only tree nuts native to the United States and are produced in several states including, Georgia, New Mexico, Texas, Oklahoma, and Arizona (1). Pecans are a high-water crop, and depending on the region, different irrigation methods are used (11). Routinely, nitrogen, phosphorus, potassium, and zinc are applied to pecans. Nitrogen is applied through irrigation, and zinc is applied through foliar application (11). Pecans are harvested mechanically by shaking trees and scraping nuts from the ground. If pecans remain on the ground for too long, nuts can become rancid and the seed coat of the nut will darken (12). During processing, pecans are conditioned in cold or hot water to soften the shells prior to cracking or shelling. After separating the inedible portions of nuts, pecans are dried to reduce the moisture content.

Iran is the leading producer of pistachios, followed by the United States (9). In the United States, about 98% of pistachio production occurs in California (13). Among the tree nuts, pistachios are the most tolerant to drought-related stress conditions. Copper, zinc, and boron are the major nutritional deficiencies in pistachios (14). The high moisture content of pistachios (40 to 50%) makes them susceptible to contamination and mechanical injury if nuts drop onto the orchard floor during harvesting. Pistachios are harvested with shake-catch mechanical harvesters, followed by placing the nuts in bins or trucks and transporting them to the huller or dehydration plants (14). Pistachios must be processed within 12 to 24 h of harvesting (13) to prevent quality issues. After the removal of hull, nuts are dried to a moisture content of 5 to 6% using a single-stage or two-stage system. Unhulled pistachios are kept in cold and low relative humidity conditions to minimize losses (0°C and <70% relative humidity) (8).

Two main varieties of walnuts are grown in the United States, English walnuts and black walnuts. Black walnuts are used as rootstocks for English walnuts (13). Virtually all walnut production in the United States occurs in California. Among the different types of walnuts, English walnuts are primarily grown in California (1). Walnuts are generally produced in areas with little to no rainfall to prevent foliar diseases, and the crop is generally dependent on irrigation for its water requirements. Like almonds, nitrogen, potassium, and zinc are the major nutrients applied to the crop (15). Walnuts are swept into windrows immediately after mechanical harvesting and placed on carts, bins, or trailers. They are transported to hullers to remove the hull, then dried at 43°C to reduce the moisture content to 8%. Walnuts can become rancid if dried at temperatures higher than 110°C (8). The drying period can range from 4 h to 2 days depending on the moisture content of the nuts. In-shell nuts can then be transported to the processing plants (8).

Peanuts are native to South America. In the United States, Georgia is the major producer of peanuts in terms of acreage as well as production (16). Peanuts (also called groundnuts), unlike tree nuts, are grown below the ground and take 4 to 5 months to reach maturity. Peanuts are usually inverted using a specialized machine before harvesting and allowed to dry in windrows in open air. They are usually harvested at approximately 35 to 50% moisture and dried to prevent aflatoxin growth (17). Too quick or too slow drying of peanuts can increase the splitting of kernels during shelling (18). The moisture content of dried-in-shell peanuts is maintained around 7 to 10% to prevent mold growth, and shelled peanuts are dried to 7% for maximum shelf life (18). Peanuts are generally not exposed to water during processing. Dirt, stones, plant debris, and other foreign materials are removed from peanuts with the help of screens and blowers. Cleaned peanuts are sized and graded to prevent crushing of kernels while shelling. They are passed through rollers to crack the pods and separate kernels. Shaker screens are used to separate shells from kernels. Following separation, kernels are graded and either packed in bags or shipped in bulk (17). The majority of the peanut crop gets processed, but there is demand for fresh peanuts as well (16).

Natural Microflora on Nuts

Both nuts and grains are low in moisture content and water activity, which prevents the growth of most microorganisms on their surface. Close proximity of nuts to soil during production (peanuts), harvesting (almonds, pecans, walnuts), and postharvest handling can influence the type and population of microorganisms present on nuts (19). Alternaria spp., Aspergillus spp., Cunninghamella spp., Cladosporium spp., Fusarium spp., Penicillium spp., Rhizopus spp., Trichoderma spp., and Verticillium spp. have been identified from tree nuts; some of these strains can produce mycotoxins (19, 20). When fungal organisms are present superficially on the surface of shells or nuts, they can be removed; however, some fungal species such as Aspergillus spp., Penicillium spp., and Rhizopus spp. have been obtained from inside almonds, brazil nuts, pistachios, and walnuts, indicating that these organisms can internalize and close contaminate in-shell nuts (19). The significant differences in the type of fungal organisms present on nuts have been observed between field-collected and store-bought almonds, brazil nuts, pistachios, and walnuts, where Aspergillus flavus, Aspergillus niger, and Rhizopus spp. were associated with store-bought tree nuts, and Penicillium spp. were associated with field-harvested tree nuts (19). Peanuts are grown beneath the soil and can be contaminated with the aflatoxin-producing fungi A. flavus and Aspergillus parasiticus. Other genera commonly associated with peanuts include Fusarium spp., Macrophomina phaseolina, Botryodiplodia theobromae, Penicillium spp., and Rhizopus spp. (21).

Fungal spores can be transmitted to nuts from air, insects, and soil. Physical, chemical, and biological factors can impact the production of aflatoxin by fungus on food (22). Environmental factors such as temperature, relative humidity, composition of gaseous compounds, and moisture content of stored nuts can influence the growth of microorganisms. Chemical factors such as the use of fungicides and insecticides can alter mold colonization and aflatoxin production (22). Insect or bird damage of nuts amplifies the chances of invasion of nuts by molds and concomitant aflatoxin production (23). Aflatoxin is a potent carcinogen and teratogen to humans and mainly affects peanuts, corn, corn seed, and tree nut crops (23). In Europe, raw and ready-to-eat almonds have been required to not to exceed 15 and 10 ppb of total aflatoxin, respectively, since 2010 (24). The stringent threshold levels of aflatoxin set for trading purposes are due to growing food safety concerns at an international level.

Enterococcus and coliform microorganisms are the predominantly identified bacteria on nut surfaces (25). The major bacterial genera identified are Streptococcus, Staphylococcus, Bacillus, Xanthomonas, Achromobacter, Pseudomonas, Micrococcus, and Brevibacterium (26, 27).

Bacterial Pathogens on Nuts

Nuts can become contaminated with foodborne pathogens at any stage of production, harvesting, processing, distribution, or consumption. Proper food safety measures should be adopted to prevent contamination issues. Salmonella is considered the target organism for dry foods, including tree nuts and peanuts, because of its long-term persistence and high heat resistance on dry foods (28–31). The presence of high levels of fat contributes to the enhanced resistance of pathogens on nuts (32). Infiltration of Salmonella into in-shell pecans is higher at 21°C and 35°C than at 4°C and −20°C (33). Infiltrated pathogens demonstrate higher thermal resistance than pathogens present on the surface (34). Other bacterial foodborne pathogens associated with dry foods, including tree nuts and peanuts, are Bacillus cereus, Clostridium botulinum, Clostridium perfringens, Cronobacter, Escherichia coli O157:H7, L. monocytogenes, and Staphylococcus aureus (32).

Preharvest Contamination Sources for Nuts

Soil

Preharvest contamination of tree nuts can occur from contaminated orchard soil (35). The 2000–2001 almond outbreak associated with Salmonella enterica serovar Enteritidis PT 30 is an example of contamination acquired from an orchard, but the exact source of contamination of orchard soil has not been identified (31, 35). A 5-year study was conducted to evaluate the persistence of S. Enteritidis PT 30 in orchard soil associated with the outbreak (31). A total of 53 (23%) Salmonella-positive samples were obtained; all isolates (100%) were identified as S. Enteritidis PT 30. Salmonella was more frequently isolated during the almond-harvesting months of August to October, indicating the potential transfer of Salmonella from orchard soil to almonds as they fall to the ground during harvest or the wide distribution of the organism through an orchard due to dust production during harvest (31). Rainfall during harvesting periods can increase the amount of soil adhered to almonds and can impact the frequency of Salmonella being isolated. The probable reasons for the long-term survival of Salmonella in orchard soil have also been investigated by researchers. Almond orchard soil type, moisture content, surface and subsurface soil temperature, and air temperature were studied to determine their influence on Salmonella survival in orchard soil (36). Almond hulls that may get wet in the orchard due to rainfall or irrigation events and temperature are significant factors influencing the survival, and potential growth, of Salmonella in almond orchard soil (36, 37). Almond hulls contain significantly more soluble sugars than almond shells (36), and the liquid generated when dry hulls get wet supports the growth of Salmonella both in the hulls themselves and in soils the liquid may leach into (36, 37). Pecan packing tissue, the tissue that is inside a pecan shell in addition to the nut, consists of tannins and polyphenols and is toxic to human pathogens, which may provide some degree of natural defense against human pathogens (38). Walnut tannins are also known to have an antibacterial effect (39). Tree nuts come in contact with soil after harvesting, whereas peanuts are grown beneath the soil surface. The presence of soilborne pathogens in peanuts is inevitable, emphasizing the need to adopt proper postharvest handling and processing practices to avoid contamination of the final product.

Animals

Repetitive mowing, application of herbicides, and grazing are some of the practices adopted by orchard growers to manage cover crops (40). Sheep, goats, and cattle are used for grazing in orchards with the aim of getting better economic returns (40). Orchard grazing was commonly practiced until the 1950s, when concerns such as overgrazing, bark damage, and pathogen contamination issues reduced this practice (40). Taking pets out for walks in orchards is also practiced in some areas (41). Animal intrusion in the orchard can lead to several food safety concerns. Foodborne pathogens harbored by animals can be deposited in orchard soil by animal feces. Research conducted in the 1970s (42) analyzed the food safety concerns associated with grazing animals in pecan orchards. Two harvest seasons, 1970 (wet) and 1971 (dry), were considered in the study. Results indicated that 6 times higher E. coli contamination of pecans was found when orchards were grazed compared to ungrazed orchards, with higher positive samples obtained in the wet year (1970) compared to the dry year (1971). The hard shells of tree nuts such as almonds, pecans, and walnuts protect their kernels from dust, discoloration, and microorganisms. When the shell was intact, E. coli could not penetrate to the inside of in-shell pecans (42). Water absorption by in-shell pecans for 48 h can lead to cracking of shells along the suture line, which can further increase the chances of E. coli invasion (42).

Nut orchards can attract several kind of wild animals including deer, hogs, mice, rabbits, squirrels, and turkeys (43). Rats and mice are considered the main cause of damage in nut and fruit orchards in the United States. Accurate and timely identification of damage from rats and mice is necessary to develop effective management programs. The use of snap traps, baits, and remote-triggered game cameras are some of the techniques used to catch rats and mice in orchards. Extensive information on the design of bait stations, bait application, and cost analysis of baiting programs is available (43). Birds can also cause considerable damage to agricultural crops. Ravens, crows, scrub jays, magpies, and black birds are some of the birds responsible for damaging nut crops (44).

Harvest

Until harvesting, tree nuts do not typically come into contact with humans, soil, or equipment, which can be critical sources of contamination. If nuts are harvested to the ground, the orchard floor should be prepared prior to harvest to ensure that there are no potential sources of contamination, such as animal feces, for nuts to be harvested into. Early splitting of pistachio shells can make the kernel more vulnerable to contamination during harvest and postharvest handling (45). Mechanical harvesting of tree nuts consists of equipment such as tree shakers, nut sweepers, vacuum harvesters, trash separators, and others. Peanuts are mechanically harvested using peanut digger and harvesting combines. The use of contaminated harvesting equipment can contaminate nuts upon contact, and care should be taken to ensure that harvesting equipment is cleaned. Mechanical harvesting should be conducted in dry weather to minimize the potential for cross-contamination. In wet weather, leaves, soil, and debris can adhere strongly to nut surfaces and can affect the cleaning process and increase the chances of contamination. Historically, almonds were harvested onto canvas tarps; these nuts had lower aerobic plate and yeast and mold counts than almonds harvested from the orchard floor (27), but this practice is no longer in use. All workers involved in harvesting activities should be trained in good health and hygiene practices.

Handling

Many nuts are mechanically dried immediately after harvesting to reduce the moisture content to less than 7% with the use of forced dry heat (8). Drying nuts to an adequate moisture level is critical to prevent microbial growth on nuts that may result in aflatoxin formation. The moisture content of the nuts should be determined at several spots in each load to ensure accurate measurement. Certain nuts such as macadamias, pecans, and walnuts are exposed to water conditions to soften the outer shell prior to shelling (6). This process is called conditioning. Almonds are exposed to water during the blanching process to remove the outer skin. Seeds such as melon, sesame, and pumpkin are also sometimes exposed to water treatment for the hulling process (46). Proper drying after water treatment is crucial for preventing the growth of microflora that are present on nuts. Beuchat and Mann (28) demonstrated the inactivation of Salmonella on inoculated in-shell pecans during conditioning treatment. Immersion-inoculated in-shell pecans immersed in chlorinated water at concentrations of 0, 100, 200, and 400 μg/ml did not reduce Salmonella populations significantly even after 24 h of treatment. Salmonella populations were significantly higher after 16 h and then after 24 h of treatment in chlorinated water. The increase in Salmonella concentration was attributed to the release of cells attached to the shell and internal tissues (47). The use of chlorine to prevent cross-contamination during conditioning or hulling may be challenging due to high organic loads present in the water. The use of hot water (80 to 95°C) was effective in reducing Salmonella cells by ca. 5 log CFU/g, but the use of higher temperatures (85 to 95°C) can compromise the organoleptic properties of nuts after 15 to 20 min. of exposure (47).

Nuts may be processed prior to consumption; the reduction of pathogens on the surface of dry foods depends on several factors such as the type of organism, process parameters, type of heat treatment, and type of food item. The time and temperature used for nut processing are critical parameters to ensure the desired lethality of foodborne pathogens. Failure to comply with the process parameters can lead to food safety issues. Dry heat is less lethal than moist heat for Salmonella on dry products. Average D values for Salmonella cocktails inoculated onto wheat, sesame seeds, walnuts, pecan nuts, pumpkin seeds, and brazil nuts and exposed to dry heat at 105°C were 132.5, 102.5, 174.6, 170.9, 235.3, and 242.1 min (48). During wet heat treatment such as almond blanching, D values for Salmonella were 2.6, 1.5, 0.75, and 0.39 min at 60, 70, 80, and 88°C water, respectively (49). Heat treatments conducted using appropriate process parameters are capable of ensuring significant and adequate reduction of pathogens on nuts.

Storage

Following harvest, nuts should be stored in a facility that is dry and provides protection from rain, groundwater drainage, rodents, and pest infestation. The storage facility should have minimum fluctuations in temperature to prevent microbial growth and insect infestation. Storage temperature should be monitored at fixed intervals. Nut handlers may store their product under controlled conditions to maintain quality. The shelf life and quality of nuts are influenced by the storage conditions (50). Almonds can be stored by nut handlers at ambient, refrigerated (4°C), or frozen (−20°C) conditions for 12 months or longer (30). The common storage temperatures (and suggested storage times) for pecans, both in-shell and kernels, include −18°C (for up to 6 to 8 years), 0°C (for about 12 to 18 months), or ambient temperature (for about 3 to 6 months) (51). Common storage temperatures (and suggested times) for peanuts, both in-shell and kernels, include −18°C (for 2 to 10 years), 1 to 5°C (for approximately 1 year), or ambient temperature (up to 6 months) (52). After processing and packaging, nuts are typically stored under ambient conditions at the retail level; consumers may store nuts under ambient, refrigerated, or freezer conditions for extended periods (53).

Salmonella survives on nut surfaces at lower temperatures (i.e., refrigeration or freezer conditions) without significant declines in populations over time, whereas slow, but significant, reductions are typically observed at ambient temperatures (29–31, 33, 54). The survival of Salmonella, E. coli O157:H7, and L. monocytogenes has been documented on almond kernels at −19, 4, 24, and 35°C (30), on peanuts at −24, 4, and 22°C (29), on pecans at −24, 4, and 22°C (29), on pistachios at −19, 4, and 24°C (30), and on walnut kernels at −20, 4, and 23°C (54) for at least 365 days. Salmonella can survive in peanut butter and peanut butter spreads for at least 168 days at 5 and 21°C (55). At −20, 4, 21, and 37°C, Salmonella can survive on pecan halves and pieces for 365 days and on in-shell pecans for 550 days (33). Salmonella survives better than E. coli O157:H7 and L. monocytogenes on the surface of nuts (29, 30, 54), making it the target organism for low-water-activity foods. Under dry conditions, Salmonella produces cell division inhibitors and accumulates compatible solutes to maintain the turgor and osmotic pressure that helps it survive in the environment (56).

Prevalence and Concentration of Pathogens on Nuts

Limited information is available on the prevalence and concentration of human pathogens on nuts. The prevalence of Salmonella on raw almond kernels, sampled over a period of 7 years, was 0.98 ± 0.32%, where 137 almond samples out of 13,972 almonds were positive for Salmonella (57–59). The concentration of Salmonella determined using a three-tube most probable number (MPN) method on raw almond kernels ranged from 0.0044 to 0.15 MPN/100 g. Out of 81 positive samples, 35 serotypes of Salmonella were identified (58). Natural Salmonella prevalence on raw in-shell almonds was 1.5% of 455 100-g samples tested (57). Using the MPM method, 44 (0.95%) out of 4,641 in-shell pecans were positive for Salmonella, and the levels obtained were 0.47 to 39 MPN/100 g with a mean of 2.4 MPN/100 g (60). A total of 31 serotypes of Salmonella were obtained from 42 Salmonella-positive pecan samples (60). The prevalence of naturally present Salmonella on raw shelled peanuts in the United States was 2.3% of 944 peanut samples (375 g each) from three crop years (61), and the corresponding concentration of Salmonella on peanuts as determined by an MPN assay was <0.030 to 2.4 MPN/g (3 to 240 MPN/100 g). Another study of raw shelled peanuts found Salmonella and enterohemorrhagic E. coli in 0.67% and 0.030% of 10,162 peanut samples (350 g each), respectively, averaged over three crop years (62); the calculated Salmonella levels were 0.74 to 5.25 MPN/350 g (0.21 to 1.5 MPN/100 g).

Processing practices reduce the microbial load on nuts. The presence of pathogens on processed ready-to-eat products is unacceptable and makes the product unfit for consumption. A survey of edible roasted nuts conducted in the U.K. retail market resulted in one S. enterica serovar Havana-contaminated pistachio sample out of a total of 727 samples (0.13%) of different nuts tested (63). Edible brazil nut kernels analyzed for the presence of Salmonella in the U.K. retail market resulted in 0.40% of 469 positive samples contaminated with S. enterica serovars Seftenberg and Tennessee at the levels of <0.010 to 0.23 MPN/g (<1 to 23 MPN/100 g) using a 10-tube MPN method (63). The same group of researchers (63) also found S. enterica serovar Anatum from a sample of mixed nuts (almond, Brazil nut, cashew, peanut, walnut) out of 329 samples tested during their survey. The levels of S. Anatum obtained from a mixed nut sample was <0.010 MPN/g (1 MPN/100g) using a 10-tube MPN method (63). A study conducted to determine the microbiological safety of edible seeds in the U.K. retail market observed Salmonella in alfalfa seeds (1.7% of 58 samples), linseeds (0.40% of 284), melon seeds (8.5% of 47), sesame seeds (1.7% of 771), and sunflower seeds (0.10% of 976), with the overall prevalence of Salmonella in edible seeds observed to be 0.60% (64). The presence of pathogens on ready-to-eat nuts is a serious concern and, if not identified in a timely manner, can result in foodborne illnesses.

Recalls and Outbreaks

Almonds, cashews, coconuts, hazelnuts, pine nuts, pecans, pistachios, and peanuts, as well as several nut products, have been associated with foodborne outbreaks and/or recalls (65, 66). The majority of these outbreaks and recalls have been associated with Salmonella. The foodborne illness cases from multiple U.S. states (67–73) and/or from other countries (35, 74–83) are highlighted in these outbreaks. Outbreaks and recalls due to E. coli O157:H7 (84, 85) and recalls due to L. monocytogenes contamination (86) are documented for some nuts and nut products. Due to the long shelf life and the long-term survival of human pathogens on nuts, most of the nut-associated outbreaks have lasted for a period of several months. The cases from an almond and peanut butter outbreak were identified over 8 to 9 months and 4 to 10 months, respectively (35, 67, 68, 71, 74). Nut outbreak investigations rarely link the outbreak strain to its original source. The 2000–2001 outbreak of salmonellosis caused by S. Enteritidis PT 30 associated with raw almonds was a rare outbreak where the investigation identified an almond orchard to be the source of contamination, but the source of contamination of the orchard soil was not identified. The S. Enteritidis PT 30 strain was subsequently isolated from the almond orchard floor over a period of 5 years (31).

Seeds have also been identified as the source of foodborne illnesses and recalls in the recent past. Sesame seed products (tahini, halva, and hummus) have been involved in seven foodborne disease outbreaks in different parts of the world since 2001 (87–91). In all the cases, the product was procured from either Turkey or Lebanon. Epidemiological investigations identified Salmonella as the causative agent in all these outbreaks. In 2014, chia seeds and chia powder were involved in a multistate outbreak of salmonellosis in the United States (92). The outbreak caused 73 infections and 8 hospitalizations in the United States and Canada (92).

GRAIN MICROBIOLOGY AND CONTAMINATION SOURCES

By definition, grains are the fruiting bodies of various grasses (93). Grains belong to the family Poaceae and are similar in structure and biochemical properties. Typically, grains are dense in energy and vary in their starch content from 50 to 80% depending upon their origin and environmental conditions. Grains include wheat, rice, barley, oats, maize, buckwheat, sorghum, millet, and mixed grains. Grains are used for food, feed, and silage production in all parts of the world (93).

Fungi Associated with Grains

Fungal pathogens are more tolerant to dry environments than bacterial pathogens and are considered a significant problem for cereal products. Air, soil, human contact, animals, insects, and pests can be possible sources of contamination of grains. Environmental conditions such as warm temperature, high humidity, rainfall, drought, sunlight, frost, and wind can lead to proliferation of fungal contaminants on grains. Contamination of cereal grains with various spoilage- and mycotoxin-producing fungi can occur prior to harvesting (94). Fungi associated with cereals can be divided into “field fungi” and “storage fungi.” Field fungi primarily consist of Alternaria, Cladosporium, Fusarium, and Helminthosporium. Field fungi infect grains in the field at high moisture content (18 to 30%), high water activity (>0.9), and high humidity conditions (90 to 100%) (95). Field fungi can cause seed discoloration, shriveling, blemishing, loss of germination, and mycotoxin production and have the potential to survive on grains for long periods of time, depending on the environmental conditions of storage (95). Storage fungi can infect and invade cereal grain after harvesting. Aspergillus, Rhizopus, Mucor, Wallemia, and Penicillium are the genera of fungi infecting grains during storage (96). Damaged grains are more prone to fungal infestation and mycotoxin production than undamaged grains (97).

Various postharvest techniques, such as physical separation of damaged kernels, separation through filtration, solvent extraction, milling, and inactivation of mycotoxins by heat can reduce the microbial contaminants from grains and enhance the product safety (94). However, the complete elimination of mycotoxins from grain is impossible to achieve (98), emphasizing the requirement to have better prevention strategies in place. Various processing techniques such as baking, cooking, brewing, roasting, and flaking have variable effects on mycotoxin concentration (98). Utilization of proper harvesting, handling, and storage practices can prevent contamination to a large extent. Lowering the moisture content of grains to 12% from 14% and maintaining low humidity conditions in the storage environment can prevent fungal growth during storage. The use of antifungal agents such as propionic acid and acetic acid has also been practiced to prevent fungal contamination of grains (96). Regulatory limits set by the U.S. FDA for mycotoxin in grains helps keep the grain supply safe for consumption in the United States.

Bacteria Associated with Grains

A wide variety of spoilage and pathogenic bacteria can grow on cereal grains, including lactic acid bacteria, coliforms, Enterococci, B. cereus, Clostridium spp., Salmonella, and S. aureus (99). Most of the microorganisms are present on the pericarp layer of the grain. Removal of the outer layer of grains can significantly reduce the microbial load. The removal of 4% of total weight of wheat using abrasive milling can reduce microbial populations by ca. 87% (100); simple agitation in a liquid medium did not result in the removal of the strongly adhered microorganisms from the surface of wheat grains (100).

As with nuts, grains can acquire contamination from soil, harvesting equipment, animal feces, or inadequate storage conditions. Postprocess handling can also cause serious food safety issues. Spore-forming bacteria can withstand the adverse processing conditions and can be carried over to the cereal products, which can lead to spoilage issues such as ropiness in bread caused by Bacillus subtilis. Bacteriostatic agents such as propionates can assist in the prevention of rope formation in bread and other quality issues (101). B. cereus is an important foodborne pathogen of concern for grains, especially rice. The bacterium is present on the outer casing of rice and can withstand high cooking temperatures and improper refrigeration conditions. Germinated spores can be killed with adequate cooking conditions. Immediate cooling of cooked rice or maintaining the cooked rice at a hot temperature is pertinent to prevent foodborne illnesses associated with B. cereus (102). C. botulinum is a soilborne spore-forming organism and can come in contact with grains during production. The growth of bacteria can be prevented by keeping the grains dry and cool. Water activity of <0.93 can prevent the production of toxin by C. botulinum (103).

Food safety recalls and outbreaks have been linked to cereal products due to pathogen contamination. In 1998, S. enterica serovar Agona was involved in the disease outbreak linked to toasted oats prepared at a Minnesota cereal manufacturing facility. Ten years later, in 2008, Malt-O-Meal unsweetened puffed rice and puffed wheat cereal prepared at the same facility was also implicated in a multistate disease outbreak caused by S. Agona (104). The original source of this outbreak was not identified; however, the pulsed-field gel electrophoresis pattern of the S. Agona strain obtained from the puffed cereal product was indistinguishable from the one obtained from toasted oats, leading to permanently shutting down that section of the facility. The presence of S. Agona in the same facility after a span of 10 years indicates the long-term persistence of this pathogen in the dry food production environment. Cronobacter is also commonly associated with dry food environments; it has been isolated from dry products such as rice seeds (105), rice starch and flour, and brown rice (106), as well as from a dry processing plant during environmental sampling (107). Cronobacter can survive and grow in infant rice cereal at various temperature conditions (108, 109). The survival of Cronobacter spp. has been documented for the period of 12 months at 4, 21, and 30°C conditions, typically observed during retail and consumer storage at home (109).

GOOD AGRICULTURAL PRACTICES FOR NUTS AND GRAINS

The use of safe practices can help in reducing the natural microbial loads of nuts. Good agricultural practices are the various farming practices that have the potential to reduce contamination in food. Specific guidelines for individual crops vary, but general guidelines recommend prevention of contamination from soil, water, animals, and humans. Various recommended practices for nuts and grain producers are as follows.

Site History

Prior use of agriculture land for animal husbandry, pasture purposes, or animal feeding operations can cause food safety issues in nuts, mainly peanuts, which are grown beneath the soil surface (110). Industrial waste dumped into the soil consists of high levels of heavy metals such as lead, arsenic, and mercury. The lack of mobility of heavy metals in plants prevents their uptake by tree nuts (110), but peanuts can be exposed to heavy metals during their growth below the ground level. Agricultural land used for nut and grain cultivation should not have a history of being an industrial dump site. Even the proximity of agricultural land to animals can lead to the contamination of nuts through water runoff from animal operations to farm land and/or air flow from livestock to farm land. Vegetative buffers can help in preventing water runoff from adjacent lands and prevent the introduction of airborne pathogens into orchard land by blocking the wind coming from nearby areas (110).

Water Source

Water is used on the farm for irrigation, for mixing pesticides and fertilizer, and for foliar application. At shelling facilities, water is used for conditioning tree nuts to soften the outer shell and facilitate the shelling process. At any step where water contacts the harvestable portion of the crop, care should be taken that the water is safe and of adequate quality for its intended use. In harvest and postharvest applications, potable water should be used, and if practical, sanitizer levels should be maintained in water to avoid cross-contamination. Contaminated water used for conditioning purposes has the potential to contaminate nuts (33). If hot water conditioning is considered as a “kill step,” proper validation of the treatment is required.

Agricultural Land Management

The orchard floor used for tree nut harvesting should be properly managed for weeds and grasses to expedite the harvesting process (111). Grazing of animals in tree nut orchards should either not be practiced or animals should be removed a few months prior to harvesting to allow manure to cake and dry. Wildlife (e.g., rodents, birds) can contaminate the nuts in the trees and should be kept out of the orchard site as much as possible. Although it may be difficult to completely eliminate grazing animals and wildlife from orchards, effective management strategies can help in minimizing the risk associated with them (43, 44), such as preharvest inspections for animal feces and its removal prior to harvest. Insect pests can also damage the nut crop and should be controlled using integrated pest management programs.

Agricultural Chemical Use

Spraying agricultural land and crops with insecticides, pesticides, and herbicides protects the crop from damage. Spraying should be conducted by trained personnel and according to the instructions provided by the manufacturer. Only Environmental Protection Agency (EPA)-registered chemicals should be used for spraying, and they should be used according to label requirements. Individuals should have personal protective equipment including gloves, masks, and spray suits to prevent exposure to pesticide. All chemicals should be stored in locked, well-ventilated rooms away from the food source. Only authorized personnel should have access to the chemical storage room.

Manure Application

Human pathogens such as E. coli O157:H7 and Salmonella reside in the gastrointestinal tract of animals such as cattle, pigs, deer, sheep, and others. Animals shed these pathogens in their feces. Improperly treated compost or manure used in the field can be a potential source of contamination for crops, mainly peanuts growing beneath the surface of the soil. Growers should be aware of contamination and environmental issues associated with using improperly treated manure on farms (112). Manure storage sites close to orchards or agricultural land can also be a source of contamination of nuts. With an inadequate slope, runoff from the storage site to the orchard during rainfall or dust due to prevailing winds can increase the chances of contamination, so manure storage sites, if present, should be contained with physical barriers.

Worker Hygiene

Workers should be trained to handle the product with care and avoid any chances of cross-contamination upon handling. Proper restroom facilities, hand-washing facilities, and drinking water facilities should be available for workers. Workers should be provided with proper sick leave policies, or management strategies should be in place preventing ill workers from coming into contact with nuts, grains, or food contact surfaces to avoid cross-contamination.

Harvesting

Every effort should be made to prevent contamination during harvesting. Harvesting equipment should be in good working order, should be kept clean, and should not introduce pathogens to nuts. Equipment and transport vehicles should be inspected for obvious dirt and debris and should be cleaned prior to use. Harvesting bins should not be used for storing or transporting toxic chemicals, fertilizers, or pesticides but should be designated for nut or grain storage only.

Plant Cleaning and Sanitation

Effective cleaning and sanitation practices are critical for product safety. Facilities handling nuts and grains where no water is used in the process should be kept dry, and specific dry cleaning and sanitizing procedures should be followed, unless there is adequate time for equipment to dry after cleaning. Cereal grain and nut processing plants can have varying amounts of oil, carbohydrate, and protein deposits on surfaces, depending on the processed product (113). Dry cleaning of dry facilities minimizes moisture conditions, which subsequently prevents the proliferation of microorganisms. Processes such as scraping, wiping, sweeping, blowing, vacuuming, purging, and flushing are used for dry cleaning purposes (113). The design of equipment can also influence the effectiveness of the cleaning and sanitation practices used. Alcohol-based sanitizers (114) are more effective in controlling microbial populations in dust than water-based sanitizers and are recommended for sanitation of dry food facilities.

Documentation and Record Keeping

Records about the history of the farmland, fertilizers, pesticide application, and worker training and information on water sources, water testing records, and harvest dates should be kept. Trace-back is an important tool to identify the exact source of food items. It plays an important role in foodborne recall and outbreak situations, where consumer health could be at risk. Adequate information about the load, variety, and farm should be recorded to track-back the product to the farm, when required.