Nitrate and Nitrite Methods of Analysis and Levels in Raw Carrots, Processed Carrots and in Selected Vegetables and Grain Products

Methods for quantitative estimation of nitrate and nitrite were compared. Levels of these ions were measured in vegetables and grain products and effects of processing on nitrate and nitrite levels in carrots were measured. These data allow more accurate estimation of ingestion levels and suggest means to reduce exposure to these ions. High performance liquid chromatography (HPLC) had better precision and recoveries than either a classical Cd-Griess method nitrate or a Griess method for nitrite. Nitrate concentration by HPLC varied greatly within and between vegetables, ranging from 1 μmol/100g in mushrooms to 5000 μmol/100g in celery and averaging 9.7 ± 4.4 μmol/100g in grains. Nitrate levels in vegetables sold as “organic” were not different (p<0.05) from conventional vegetables. No nitrite was detected in either vegetables or grains. Nitrate was unevenly distributed in carrots with the core having the most. Storage of carrots at -18°C for 10 wk did not alter nitrate levels and no nitrite developed. Fifty-seven percent of nitrate was leached into cooking liquid when frozen carrots were boiled. Thirty-two percent of nitrate was lost during canning and 47% of the remainder was in the liquid. No nitrite developed during 10 wk of canned storage.     

Evidence that ingested nitrate and nitrite are beneficial to health

The literature was reviewed to determine whether ingested nitrate or nitrite may be detrimental or beneficial to human health. Nitrate is ingested when vegetables are consumed. Nitrite, nitrate's metabolite, has a long history of use as a food additive, particularly in cured meat products. Nitrite has been a valuable antibotulinal agent in cured meats and may offer some protection from other pathogens in these products as well. Nitrite's use in food has been clouded by suspicions that nitrite could react with amines in the gastric acid and form carcinogenic nitrosamines, leading to various cancers. Nitrate's safety has also been questioned, particularly with regard to several cancers. Recently, and for related reasons, nitrite became a suspected developmental toxicant. A substantial body of epidemiological evidence and evidence from chronic feeding studies conducted by the National Toxicology Program refute the suspicions of detrimental effects. Recent studies demonstrate that nitrite, upon its ingestion and mixture with gastric acid, is a potent bacteriostatic and/or bactericidal agent and that ingested nitrate is responsible for much of the ingested nitrite. Acidified nitrite has been shown to be bactericidal for gastrointestinal, oral, and skin pathogenic bacteria. Although these are in vitro studies, the possibility is raised that nitrite, in synergy with acid in the stomach, mouth, or skin, may be an element of innate immunity.     

Intake and risk assessment of nitrate and nitrite from New Zealand foods and drinking water

Exposure to excess nitrite is a potential health risk for humans. One hundred meat and processed foods and 100 vegetable samples purchased from New Zealand retail outlets were prepared as for consumption and analysed for nitrite and nitrate concentration using a standard, validated methodology. Nitrate concentrations ranged from less than the limit of detection (LOD = 5 mg kg^-1) in cheddar cheese and cream cheese-based dips to 3420 mg kg^-1 in lettuce. Nitrite was detected in half the processed foods and meats analysed (levels up to 119 mg kg^-1), but detected in only one vegetable sample above the LOD (broccoli at 27 mg kg^-1 nitrite). Concentration data were combined with 24 h dietary recall information to generate 4398 individual adult daily exposure scenarios for exogenous nitrite and nitrate including a contribution from water assessed from 1021 drinking water samples. The mean adult daily intake of exogenous nitrate and nitrite from food and water combined was 16 and 13% of the Acceptable Daily Intake (ADI), respectively, and therefore should not pose a health risk for the average consumer. A maximally exposed New Zealand adult is estimated to have an intake of up to seven times the ADI for nitrate. When the endogenous conversion of nitrate to nitrite is taken into account, approximately 10% of people with an average rate of conversion and half of all people with a high rate of conversion are estimated to exceed the ADI. Either the ADI is inappropriate and needs to be re-evaluated, or those individuals who have a high rate of conversion of nitrate to nitrite are at risk to adverse effects of nitrite exposure.     

Nitrate and nitrite quantification from cured meat and vegetables and their estimated dietary intake in Australians

High dietary nitrate and nitrite intake may increase the risk of gastro-intestinal cancers due to the in vivo formation of carcinogenic chemicals known as N-nitroso compounds. Water and leafy vegetables are natural sources of dietary nitrate, whereas cured meats are the major sources of dietary nitrite. This paper describes a simple and fast analytical method for determining nitrate and nitrite contents in vegetables and meat, using reversed-phase HPLC-UV. The linearity R² value was >0.998 for the anions. The limits of quantification for nitrite and nitrate were 5.0 and 2.5 mg/kg, respectively. This method is applicable for both leafy vegetable and meat samples. A range of vegetables was tested, which contained <23 mg/kg nitrite, but as much as 5000 mg/kg of nitrate. In cured and fresh meat samples, nitrate content ranged from 3.7 to 139.5 mg/kg, and nitrite content ranged from 3.7 to 86.7 mg/kg. These were below the regulatory limits set by food standards Australia and New Zealand (FSANZ). Based on the average consumption of these vegetables and cured meat in Australia, the estimated dietary intake for nitrate and nitrite for Australians were 267 and 5.3 mg/adult/day, respectively.     

Use of natural ingredients to control growth of Clostridium perfringens in naturally cured frankfurters and hams

A major concern for processed meats marketed as natural/organic is that they do not contain nitrite in concentrations known to be most effective for inhibiting foodborne pathogens. Supplemental treatments to increase the level and consistency of antimicrobial protection in these products may be important to provide consumers with the degree of safety that they have come to expect from conventionally cured meats. Therefore, the objective of this study was to identify and test ingredients that might improve processed meat product safety without altering their natural/organic status. Eight treatments of hams and frankfurters were prepared: (A) uncured control (typical ingredients except nitrite and nitrate); (B) conventionally cured control (erythorbate, nitrite, and a lactate-diacetate blend); (C) natural nitrate cure (including starter culture containing Staphylococcus carnosus); (D) natural nitrate cure (culture and natural antimicrobial A containing a vinegar, lemon, and cherry powder blend); (E) natural nitrate cure (culture and antimicrobial B containing a cultured sugar and vinegar blend); (F) natural nitrite cure without additional antimicrobials; (G) natural nitrite cure with natural antimicrobial A; and (H) natural nitrite cure with antimicrobial B. For the hams, treatments C, D, E, and H impacted growth of Clostridium perfringens to the same extent (P < 0.05) as the conventionally cured control (approximately 2 log less growth over time than uncured control). For frankfurters, treatments D, G, and H had an effect (approximately 1 log) on growth equivalent to that of the conventionally cured control (P < 0.05). These results suggest that natural/organic cured meats have more potential for pathogen growth than conventionally cured products, but supplemental natural ingredients offer safety improvement.     

Survival and growth of Clostridium perfringens in commercial no-nitrate-or-nitrite-added (natural and organic) frankfurters, hams, and bacon

The popularity of "preservative-free" foods among consumers has stimulated rapid growth of processed meats manufactured without sodium nitrite. The objective of this study was to quantify the potential for Clostridium perfringens growth in commercially available processed meats manufactured without the direct addition of nitrite or nitrate. Commercial brands of naturally cured, no-nitrate-or-nitrite-added frankfurters (10 samples), hams (7 samples), and bacon (9 samples) were obtained from retail stores and challenged with a three-strain inoculation (5 log CFU/g) of C. perfringens. Reduced inhibition (P < 0.05) was observed in seven brands of frankfurters, six brands of hams, and four brands of bacon when compared with each respective sodium nitrite-added control. In naturally cured and truly uncured commercial frankfurters, growth over time was approximately 4.7 log, while conventionally cured frankfurters exhibited growth at 1.7 log. Naturally cured ham and bacon products exhibited growth at 4.8 and 3.4 log, respectively, while their conventionally cured counterparts exhibited growth at 2.6 and 2.3 log, respectively. These products also demonstrated variation in growth response. The results indicate that commercially available natural/organic naturally cured meats have more potential for growth of this pathogen than do conventionally cured products. Natural and organic processed meats may require additional protective measures in order to consistently provide the level of safety from bacterial pathogens achieved by conventionally cured meat products, and which is expected by consumers.     

Use of natural antimicrobials to improve the control of Listeria monocytogenes in a cured cooked meat model system

Concern about nitrite in processed meats has increased consumer demand for natural products manufactured without nitrite or nitrate. Studies on commercial meat products labeled as "Uncured" and "No-Nitrite-or-Nitrate-Added" have shown less control of nitrite in these products and greater potential growth of bacterial pathogens. To improve the safety of the "naturally cured" meats, several natural ingredients were studied in a cured cooked meat model system (80:20 pork, 10% water, 2% salt, and 150 or 50 ppm ingoing sodium nitrite) that closely resembled commercial frankfurters to determine their inhibitory effect on Listeria monocytogenes. Results showed that cranberry powder at 1%, 2% and 3% resulted in 2-4 log cfu/g less growth of L. monocytogenes compared to the control with nitrite alone (P<0.05). Other natural compounds, such as cherry powder, lime powder and grape seed extract, also provided measureable inhibition to L. monocytogenes when combined with cranberry powder (P<0.05).     

Spectrophotometric Determination of Nitrite and Nitrate in Cured Meat by Sequential Injection Analysis

ABSTRACT: A robust, automated, labor-saving, accurate, and economical sequential injection system was developed for simultaneous determination of nitrite and nitrate in cured meat samples, based on the Shinn reaction. Nitrite is coupled and diazotized with sulfanilamide and N-(1-naphtyl)-ethylenediamine dihydrochloride, to form a colored compound that absorbs at 538 nm. Nitrate is previously in-line reduced to nitrite in a copperized cadmium column and measured as nitrite. The solutions' aspiration sequence, the influence of reagent and buffer concentrations, the manifold parameters, and the characteristics of the reducing column were studied. Nitrite and nitrate can be determined within the 0.030 to 1.22 of N-NO₂− and 0.034 to 3.95 mg/L of N-NO₃− ranges, respectively, at a sampling rate of 9/h. Detection limits of 9 μg/L of N for nitrite and 9 μg/L of N for nitrate were obtained, and the conversion rate of nitrate to nitrite was 100.6%± 1.8%. The results were in good agreement with those obtained by the reference methods, with relative standard deviations (r.s.d.) better than 3.70% for nitrites and 2.42% for nitrates.     

Time-dependent depletion of nitrite in pork/beef and chicken meat products and its effect on nitrite intake estimation

The food additive nitrite (E249, E250) is commonly used in meat curing as a food preservation method. Because of potential negative health effects of nitrite, its use is strictly regulated. In an earlier study we have shown that the calculated intake of nitrite in children can exceed the acceptable daily intake (ADI) when conversion from dietary nitrate to nitrite is included. This study examined time-dependent changes in nitrite levels in four Swedish meat products frequently eaten by children: pork/beef sausage, liver paté and two types of chicken sausage, and how the production process, storage and also boiling (e.g., simmering in salted water) and frying affect the initial added nitrite level. The results showed a steep decrease in nitrite level between the point of addition to the product and the first sampling of the product 24 h later. After this time, residual nitrite levels continued to decrease, but much more slowly, until the recommended use-by date. Interestingly, this continuing decrease in nitrite was much smaller in the chicken products than in the pork/beef products. In a pilot study on pork/beef sausage, we found no effects of boiling on residual nitrite levels, but frying decreased nitrite levels by 50%. In scenarios of time-dependent depletion of nitrite using the data obtained for sausages to represent all cured meat products and including conversion from dietary nitrate, calculated nitrite intake in 4-year-old children generally exceeded the ADI. Moreover, the actual intake of nitrite from cured meat is dependent on the type of meat source, with a higher residual nitrite levels in chicken products compared with pork/beef products. This may result in increased nitrite exposure among consumers shifting their consumption pattern of processed meats from red to white meat products.     

Exposure estimates of nitrite and nitrate from consumption of cured meat products by the U.S. population

The dietary exposures of nitrite and nitrate from consumption of cured meat products were estimated for the U.S. population aged 2 years and older, and children aged 2 to 5 years, using both 2-day food consumption data from the publicly available combined 2009–2012 National Health and Nutrition Examination Survey (NHANES) and 10–14-day food consumption data from the 2009 and 2012 NPD Group, Inc. National Eating Trends-Nutrient Intake database (NPD NET-NID), and residual nitrite and nitrate levels in cured meat products available from the recent American Meat Institute Foundation/National Pork Board (AMIF/NPB) national market survey of the nitrite and nitrate levels in cured meat products in the U.S.A. The dietary exposure for consumers of cured meat products (eaters-only) was estimated at the mean and 90th percentile for three exposure scenarios: low exposure, average exposure, and high exposure, to account for the range in the amount of nitrite and nitrate in a given cured meat product category. In addition, a cumulative exposure that takes into account all cured meat product categories containing nitrite and nitrate was determined, and the relative percent contribution of each cured meat product category to the cumulative exposure was estimated. Cured, cooked sausages and whole-muscle brine-cured products were the two major contributing categories to dietary exposure of nitrite and nitrate for both U.S. population aged 2 years and older and children aged 2–5 years.     

市售鲜肉食品的贮存与质量变化的研究

对鲜肉中的蛋白质,亚硝酸盐及硝酸盐分别用凯氏法,N－1－萘基乙二胺分光光度法和镉柱还原分光光度法进行了测定。在48h内,在常温29℃下,鲜肉中蛋白质明显下降,大头鱼降低72％,猪肉降低74.1％,草鱼降低64.5％,鸡肉降低48.5％。而亚硝酸却又显著升高,其升高的倍数依次分别为200,40,60和80。若在4℃冷藏,则蛋白质的降低和亚硝酸盐的升高速度,将大为减少,蛋白质只降低35％～6.0％,亚硝酸盐只升高14～54倍。     

Color Stability of Radappertized Cured Meat

Porcine semimembranosus muscles were cured (with different levels of nitrite, nitrate, and their combination), smoked, and irradiated with 3.2 Mrad gamma radiation at ?40°C. Subjective studies by a trained panel, objective studies of total color difference (calculated from tristimulus values), and behavior of residual nitrite and nitrate show that radiation alters the pink color of cured meats (radiation- induced fading). Contrary to prior reports, nitrate affects neither color development nor post-irradiation fading. Color of nitrate- cured samples, like uncured samples, become bright red when irradiated, due to reduction of globin myohemichromogen (indistinguishable from pink color of cured meat), and therefore are more desirable.     

替代亚硝酸盐生产安全腌肉制品的研究现状

硝酸盐和亚硝酸盐在腌肉制品加工中同时起着发色、抑菌、抗氧化和提高风味的作用,但硝酸盐和亚硝酸盐与肉中的二级胺反应会形成致癌物质N-亚硝胺,所以寻求一种更加安全有效的硝酸盐和亚硝酸盐替代方式,在实际生产中具有重要意义。本文就无硝腌肉制品在亚硝酸盐发色作用、抑菌作用和抗氧化作用的替代物方面开展的研究工作进行综述,提出了天然腌制的概念,重点介绍目前国内外利用蔬菜替代亚硝酸盐的方法,包括将蔬菜粉(或汁)中的硝酸盐用硝酸盐还原菌转化为亚硝酸盐后加入腌肉制品中(先发酵法)和将蔬菜粉(或汁)加入肉馅中再发酵(后发酵法)两种生产方式,并对目前的研究现状、进展、存在问题及今后发展方向进行了论述,以期为生产安全腌肉制品提供借鉴。     

The determination of nitrate and nitrite in cured meat by HPLC/UV

A rapid strong anion exchange HPLC/UV procedure has been developed for the determination of nitrate and nitrite in a wide variety of cured meats. The accuracy of this technique has been confirmed by the good agreement achieved with the existing British Standard colorimetric method. The applicability and repeatability of the procedure has been established in a survey of over 200 samples. The agreement between duplicate determinations and their respective means averaged +/- 3.4% for nitrite and +/- 4.3% for nitrate as defined by the term [(a - b)/(a + b)] X 100% where a and b are the repeat determination values.     

Inhibition of Listeria monocytogenes using natural antimicrobials in no-nitrate-or-nitrite-added ham

Consumer demand for foods manufactured without the direct addition of chemical preservatives, such as sodium nitrite and organic acid salts, has resulted in a unique class of "naturally" cured meat products. Formulation with a natural nitrate source and nitrate-reducing bacteria results in naturally cured processed meats that possess traits similar to conventionally cured meats. However, previous research has shown that the naturally cured products are more susceptible to pathogen growth. This study evaluated Listeria monocytogenes growth on ham manufactured with natural curing methods and with commercially available clean-label antimicrobials (cultured sugar and vinegar blend; lemon, cherry, and vinegar powder blend) and assessed impacts on physicochemical characteristics of the product. Hams made with either of the antimicrobials supported L. monocytogenes growth similar to that in the traditionally cured control (P > 0.05). Hams made with prefermented celery juice powder had the lowest residual nitrite concentrations (P < 0.05), and when no antimicrobial was added, L. monocytogenes growth was similar to that of the uncured control (P > 0.05). Aside from residual nitrite and nitrate concentrations, few physicochemical differences were identified. These findings show that ham can be produced with natural curing methods and antimicrobials to provide similar L. monocytogenes inhibition and physicochemical traits as in traditionally cured ham.     

不同氮素形态和用量对烤烟硝酸盐和亚硝酸盐含量的影响

研究了不同氮素形态和用量对烤烟硝态氮含量的影响.结果表明:鲜烟叶硝酸盐含量随硝态氮比例和施氮量的增加而呈现出上升的趋势,其含量相应低于烤后烟叶,并与烤后烟叶的硝酸盐和亚硝酸盐含量之间均存在着极显著的正相关关系;烤后烟叶硝态氮含量与硝态氮比例和施氮量之间均呈显著或极显著正相关,硝酸盐和亚硝酸盐含量的变化规律在不同部位的烟叶间表现一致,二者之间的相关也达到极显著水平,但中部叶的硝态氮含量明显高于上部叶.此外,烤后烟叶烟碱和总氮含量与施氮量均呈极显著正相关,与硝态氮比例的相关则未达显著水平.     

The microbiological role of nitrite and nitrate

In unheated products nitrite, together with sodium chloride and the pH value, contributes to the selection of the bacteria which grow during storage. Nitrate per se is generally believed to serve only as a reservoir for nitrite, but the commercial use of nitrate-free cover brines in the Wiltshire bacon industry shows that such a reservoir is not always essential. Nitrate sometimes reduced the growth rate of bacteria in experimental Wiltshire collar bacon, but was of no benefit in back bacon. The clostridia occurring naturally in the bacon grew to higher numbers in collar cured without nitrate than that cured with nitrate. Clostridium botlinum (types A and B) was detected in these bacons, but did not grow in the bacon. In heated products the growth of surviving bacteria is controlled by the interaction of several factors including pH, sodium chloride, storage temperature and sodium nitrite or a substance derived from it upon heating. Further experiments are warranted to investigate the effects of dextrose, nitrate, ascorbate and polyphosphate.     

Determination of nitrite and nitrate levels in meat and vegetable products by high performance liquid chromatography

A range of fresh and processed meat and vegetable products was analysed by h.p.l.c. for nitrite and nitrate contents. Satisfactory results were obtained for vegetable products and most meats, but some meat products were subject to matrix interference. Sucrose, sulphate and phosphate did not affect results. Added sodium chloride decreased retention time of nitrite and increased apparent levels of both nitrite and nitrate. Ascorbic and erythorbic acids decreased apparent nitrite levels and correspondingly increased apparent nitrate levels.     

施磷对烤烟硝酸盐和亚硝酸盐含量的影响

研究了磷肥用量对烟叶硝态氮含量的影响。结果表明 :在一定的施磷范围内 ,硝酸盐和亚硝酸盐含量均随施磷量的增加而呈现出下降的趋势 ;鲜烟叶的硝酸盐含量明显低于烤后烟叶 ,且与烤后烟叶的硝酸盐和亚硝酸盐含量均存在着极显著的正相关关系 ;硝酸盐和亚硝酸盐含量的变化在不同部位的烟叶间表现一致 ,但中部叶的 (亚 )硝酸盐含量高于上部叶。此外 ,施磷量与烟碱、总氮和蛋白质等其它含氮组分之间的相关关系不显著。     

Modification of the ion exchange HPLC procedure for the detection of nitrate and nitrite in dairy products

Existing ion exchange HPLC methodology for nitrate and nitrite analysis in cured meat products suffers from high analyte variability at low concentrations and also chromatographic interference by artifacts in some other foods, such as dairy products. An investigation into the sources of variability has shown that both the cyclohexyl solid phase extraction cartridge and the glass fibre filter used in the original method can introduce artifacts which interfere with the determination of the nitrate in foodstuffs. We have also found that the use of a graphitized solid phase extraction cartridge used in tandem with the cyclohexyl solid phase extraction cartridge removed the artifacts from the chromatograph of dairy products that co-eluted with nitrite and nitrate. Values for the nitrite and nitrate content of dairy products were obtained by the HPLC procedure using these two solid phase extraction cartridges and the values obtained were in close agreement with those obtained by cadmium column reduction and colorimetry.