Polyamines in foods: development of a food database

Database development and definition

An extensive literature search of databases was conducted using the keywords polyamines, food, food analysis, diet, and the individual names for the three polyamines selected for addition to the database: putrescine, spermine, and spermidine. This search was done using PubMed, web of Science, and SciFinder Scholar that cover both Medline and CAPLUS databases. Both Medical Subject Headings (MeSH) and text word searching were used to locate any published laboratory data for polyamines contents in foods. Papers published between 1986 and 2009 were selected.

When a single reference reported multiple analyses for the same food group, or if values for an individual food item were obtained from several references, the mean for all given values was calculated. However, when values reported from different references were extremely different, the median was taken to minimize the influence of extreme values. The average polyamine content for each food was then entered in the Dietist XP. All collected information on polyamine content in different foods was gathered into an Excel file where they were classified into food groups.

Values for polyamines content in some of the studies have been reported in nmol/g of food. These values were converted into mg/kg based on the appropriate equation (mol = mass/molecular weight). The molecular weights for putrescine, spermine, and spermidine are 88.15 g/mol, 145.25 g/mol, and 202.34 g/mol, respectively. In addition, portion size was also considered due to the fact that portion size and frequency of consumption of these foods vary considerably, which means that a normal food frequency questionnaire would not necessarily detect intake of products with high amounts of polyamines; for example, sauerkraut or well-fermented cheese (26).

Portion size estimation

The Swedish National Food Administration (Livsmedelsverket) has estimated the portion size for 1,334 foods. All portions in grams are available in the Swedish Food Database where the units in grams or deciliters can be found for most of the food available.

In the Dietist XP software (version 3.0, 2007) where the polyamine contents are entered, the weight of the food per portion size, the weight in grams per deciliter for liquid food, and the amounts in grams for each spoon or cup are all collected from the same data that is available in the Swedish Food Database from Livsmedelsverket, 2001. Thereby the content of each polyamine in foods per portion can also be estimated using the Dietist XP software.

Sampling

All samples were collected and analyzed between October and December 2009. Samples of cow's milk (0.5%, 1.5%, 3%, and 4.5% fat), yogurt, and sour milk were purchased from the local market 1 day before the analysis and at least 1 day after production. The best before dates were in 7 days for milk, 4 and 2 weeks for yogurt and sour milk, respectively. Different types of cheese with low and high fat content, varied between short- and long-term storage were also purchased (at least 2 months before best before date). The brands of all these products were representative for the Swedish mostly purchased dairy products, with the exception of Gamle Ole, the Danish cheese that was included in the analysis due to its long fermentation period (52 weeks). Only 4.5% fat milk and mature cheese were purchased from a different local market. Each sample was kept in the refrigerator at 4°C until the next day, which was the same degree of refrigerating at the market. For each dairy product, five samples (packages) of the same type of product were subjected to replicate analysis.

Chemicals

Polyamine standards (putrescine, spermidine, and spermine), internal standard (1,7-diaminoheptane), perchloric acid 70%, benzoyl chloride 99%, and acetonitrile 99.9% analytical grade were all purchased from Sigma-Aldrich (Chemie GmbH, Germany). Diethyl ether was obtained from Scharlau Chemie S.A, Spain.

Analytical method

For milk and yogurt, polyamine extraction was done by adding 3 ml of 0.6 N perchloric acid to 5 ml of the sample, spiked with 2 µM internal standard (1,7-diaminoheptane). For all types of cheese, 10 g of the sample was also spiked with a known amount of internal standard before the mixture was homogenized as described by Novella-Rodriguez et al. (33). The mixture was kept at 4°C for 1 h before centrifugation at 30,000g 10 min at 4°C to separate the protein phase from the acidic extract phase including the polyamines. As previously detailed in methods for determination of polyamines using precolumn derivatization (35), the polyamine derivatization procedure was done by treating the acidic extract of polyamines with small amounts of 2N NaOH to keep the pH above 13. The mixture was then subjected to the derivatization procedure by adding 10 µl of benzoyl chloride prior to polyamine extraction by diethyl ether (twice) and concentration by evaporation with nitrogen gas. The residue (benzoylated polyamines) was dissolved in 1 ml of 38% acetonitrile in water (the same solvent that was used as mobile phase). The resulting solution was then filtered using a syringe filter (GF Millipore MA, USA) and an aliquot of 50 µl of each sample was automatically injected onto a High Performance Liquid Chromatography (HPLC, Waters 2690) equipped with a Nova-Pak C₁₈ column (15×3.9 mm) and Waters UV detector 996. Data acquisition was accomplished with Millennium³² Version 3.0 system. The entire HPLC run was under isocratic elution with a flow rate of 1 ml/min.

Benzoylated polyamines were detected by UV absorption at 198 nm, as it has been shown to increase the absorbance by ca. 50 times when acetonitrile is used as solvent (36). Polyamine identification was based on comparison between the retention times of polyamine standards. These were prepared at known concentrations from a stock solution and subjected to the same derivatization and extraction procedures. The detection limits were lower than 0.1 mg/kg for polyamines. Based on polyamine analysis in 10 runs of replicate samples, the inter-assay coefficient of variation (CV) was 8.3–8.8%.

Data analysis

Means and standard deviations of dietary polyamine content of Swedish dairy products as well as the coefficient of variation of polyamines were calculated using SPSS version 17.0, 2008, SPSS Inc.

Polyamine in foods from the database

The literature search for any published values for putrescine, spermidine, and spermine provided data on polyamine content for more than 250 foods. The contents of polyamines in these foods are shown in Table 1 with references (2, 4–6, 15–17, 22, 23, 31–33, 37–56). Fruits (like oranges), fruit juices (orange juice and grapefruit juice), sauerkraut, cheddar cheeses, cod roe, soy sauce, and soy miso were high in putrescine. Spermidine content was high in dry soy bean, chicken liver, green peas, corn, shell fish, and blue cheese. A high content of spermine was found in most of the meat products (like sausages, pork, chicken, and turkey), some vegetables (like pumpkin), and cheese. This polyamine, on the other hand, was found in low quantities in other types of foods and frequently reported as not detected.

Based on the Dietist XP and portion size, the top 12 foods having the highest amounts of each polyamine in mg per portion were listed in Table 2. Polyamine contents according to food groups are shown in Fig. 1. The group of cheese was the highest source of spermidine, while the fruits group was the highest in putrescine. Both food groups were the most important sources of total polyamine.

Fig. 1

Mean polyamine (putrescine, spermidine, and spermine) content in each food group according to the developed database. Number of foods included in each group: fruits (29), vegetables (46), bread (14), meat (55), cheese (19), and potato (6).

Polyamine analysis of dairy products

Table 3 shows the results from our laboratory analysis of typical Swedish dairy foods. The low fat milk and sour milk had the highest total polyamine contents, whereas the yogurt had the lowest contents. Sour milk had the highest mean putrescine contents. Total polyamine level was higher in cheese with long maturation than other types. Unlike the milk, cheese that differs in fat percentage still had similar values of polyamines.

Polyamine database

Polyamine content varies noticeably in food. Data obtained from different studies reflect this variation. Using different techniques for measuring polyamines, in addition to the other influencing factors such as storage periods and conditions as well as cooking method, might also play a role in affecting the resulting database (5, 57). These different analytical methods showed some variation in polyamine contents in the same type of food. As shown in Table 1, putrescine in tomato varied from 1.2 to 122 mg/kg in three different studies (4, 22, 38). In addition, putrescine in sausage was another example of this variation not only between studies that have even used the same method (16, 22) but also varied in the same study. For example, the large variation in putrescine content in fermented Spanish sausage is explained by the metabolism of fermentative bacteria and the period of fermentation process (43). Similarly, the tremendous variation in putrescine concentration in 121 samples of sauerkraut (2.8–529 mg/kg) may be explained by the different microflora during spontaneous fermentation (52). However, values for polyamines in the main food sources such as putrescine in oranges, spermidine in Japanese and Norwegian blue cheese, and spermine in raw lean beef were similar across studies (4, 6, 16, 22).

Fermented soy bean has been reported to have considerable amounts of polyamines. Polyamines in fermented soy bean are considered to be formed by the action of lactic micro flora that is active during the fermentation process (58–60).

In Table 2, cooked soy bean and cow's liver were the highest sources for spermidine and spermine, respectively, whereas for putrescine, sauerkraut appeared to be the third important source due to its lower portion size than grape fruits and oranges. These figures differ slightly from an American study where the top 10 foods with the highest polyamine contents were reported (26). Fresh or canned corn and green pea soup had the highest spermidine and spermine, respectively. This difference could be due to the fact that the mean values for each polyamine that were estimated in their food database differ slightly from ours and also due to difference in portion size estimates.

Polyamine in dairy products

Our laboratory results showed that Swedish cheese has higher polyamine content than milk. This is natural since cheese is essentially concentrated milk. Such a finding was in accordance with other hard cheese variants from literature (16, 33, 50). The values for the mean total polyamine content, particularly putrescine and spermine in selected Swedish mature cheese were comparable to those in Spanish hard cheese (33). However, there were still reported variations in polyamine content in cheese between the studies (4, 16, 33, 38). This could be due to differences in the fermentation process and the starter culture employed in the cheese production (16, 58, 61).

As reported for other studies on polyamine contents in cheeses (4, 16, 22, 50), the putrescine content varied between matured and unmatured cheese. The high content of putrescine in matured cheese is expected to be a result of the fermentation process. A previous study showed that lactobacillus acidophilus, Streptococcus faecium, and Lactobacillus casei, which were isolated from fermented cheese, were responsible for putrescine production (61). In addition, using pasteurized milk and mesophilic or thermophilic starter bacteria favored the formation of polyamine in cheese (60). Spermine and spermidine contents were higher in Herrgårdsost, a common Swedish short maturation cheese, independent of fat content (17 and 28% fat). Herrgårdsost is made by introducing bacterial starters to pasteurized milk including lactic acid bacteria, which acidify the milk. This bacterial process besides the short maturation would explain the higher putrescine contents in Herrgårdost than in Prästost. In Prästost with 28% fat, spermine was detected only in one sample out of five. This polyamine was twice as much the content in 31% Prästost. This difference in spermine within the same type of cheese can be explained by polyamines variability not only between different types of cheese but also within the same type, which could be attributed to difference in the manufacturing process (50).

In milk, the lower the fat content, the higher the polyamine content. It has been previously reported that high fats and protein proportions might hinder polyamine extraction (33). Therefore, such effect has been considered in our method by careful polyamine extraction. Moreover, putrescine concentration in milk, particularly low fat milk, was about 10 times higher than the concentrations that were reported previously (22). This variation might be explained by the effect of milk processing on the levels of polyamines; however, when non-homogenized high fat milk was analyzed for its polyamine content, lower polyamine values were detected. Another explanation for the variation within and between studies could be the effect of seasonal variations on milk composition, storage conditions, and environmental influences and the different techniques used for polyamine determination (3, 48). The very low polyamine content in yogurt was in agreement with the contents reported from other studies (16, 56).

Limitations of the study

We did not have a large sample size when it comes to our own analyses of dairy products. However, the dairy products selected were the most commonly purchased brands in Sweden and the variation in the results from our chosen sensitive analytical method was minor.