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Iron fortification of Chinese soy sauce

Yau-tian Dai
Department of Nutrition and Food Hygiene, School of Public Health, Beijing Medical College,
Beijing, People's Republic of China

INTRODUCTION

Iron deficiency anaemia is still one of the most common nutritional deficiency diseases in the world, existing even in countries whose level of economic development has eradicated other nutritional deficiency illnesses. Iron deficiency is mainly prevalent among infants, pregnant women, and women of child-bearing age. These groups are vulnerable because of their high physiological requirements.

Iron deficiency can occur as a consequence of pathological losses of iron from the body or of physiological requirements for iron beyond the amount that is absorbed from foods. A negative iron balance results in a progression of events, going from a reduction in the body's iron deposits to iron deficiency anaemia (1).

Recently, many reports on iron deficiency have focused on tissue effects beyond the limitation of the synthesis of haemoglobin. These include alterations in the metabolism of muscle (2) and the brain (3) and in resistance to infection (4).

The most important aetiology of iron deficiency in developing countries is, rather than inadequate intake, poor absorption or bioavailability of iron from the diet. Iron in these diets comes predominantly or totally from cereals and vegetables, i.e., it is non-haem iron.

Results of many isotopic studies with intrinsically and extrinsically tagged foods have shown a very marked variation in the absorption of iron, depending on the amount of iron ingested, the nature of food iron, and the components of the diet. These studies determined the existence of two pools of iron in the intestinal tract concerned with absorption: the haem iron pool and the non-haem iron pool.

Haem iron, derived mainly from haemoglobin and myoglobin, is abosrbed intact as iron porphyrin, and is liberated within the parietal cells of the intestinal mucosa (5-8).

Iron absorption from this pool is higher than the absorption from the non-haem iron pool and is not affected by vegetables or ascorbic acid (9).

The non-haem iron comes from vegetables, cereals, and eggs, as well as from the non-haem iron in beef, chicken, fish, and in supplementary soluble iron salts (5, 10, 11). In this pool, iron absorption is modified not only by factors typical of the intestinal lumen, but also by food and other compounds that are ingested simultaneously. It is well known that beef, pork, chicken, liver, and fish enhance non-haem iron absorption, but milk, eggs, and cheese (11, 12) decrease the bioavailability of non-haem iron. Other substances, such as tannin in tea, phosphatides in the yolk of egg, calcium and phosphate salts, EDTA and some antacids, if present in significant amounts, eliminate non-haem iron absorption. Ascorbic acid, free or contained in fruits and vegetables, can enhance iron absorption about two to four-fold (12-14).

Recent studies have shown that non-haem iron in a single food can be uniformly labeled by an inorganic radio-iron tracer added to the food, probably by a very rapid isotopic exchange between the tracer and the native food iron within a common pool of non-haem iron The fact that non-haem iron in a great number of foods studied can be uniformly labeled by an extrinsic tag method makes It possible to perform valid measurements of the absorption of non-haem iron from individual foods and composite meals, dispensing with the need to prepare biosynthetic, intrinsically labeled foods with radioactive iron.

International experts have recommended iron fortification of food as the best method to prevent iron deficiency anaemia in the world. Different foods have been used as fortified vehicles in different countries and areas. Infant formulas (15), powdered milk (16), cereals (17, 18), fish sauce (19), sugar (20, 21), and salt (22, 23) have been fortified with varying degrees of success; some are still under study.

In China, iron deficiency anaemia is an important public health problem, especially in infants and pregnant women. It is very difficult to obtain accurate data on its prevalence, but it was estimated to range from 15 per cent in preschool children to 30 per cent in infants in an urban area in 1979 (Yau-tian Dai, unpublished observations from Nutritional Surveys on Preschool Children, Beijing Medical College). In some other areas of China the prevalence of anaemia may be higher than this figure, because a low intake of iron from animal foods in Chinese diets Is very common. Given this situation, it is important to attempt iron fortification of a common food vehicle to prevent anaemia. Chinese soy sauce may be the ideal vehicle for iron fortification.

Soy sauce is a dark brown liquid condiment that has been used in China for thousands of years. It is a universally and dally consumed product in every Chinese family. It is added during cooking, especially to pork, fish, chicken, and some vegetables. In China, soy sauce is centrally processed, and 15 to 20 ml of soy sauce are consumed per person per day, with the exception of infants.

Several characteristics make soy sauce a promising vehicle for iron:

- It is a liquid, so it should be easy to obtain uniform distribution of the fortificant.
- It has a dark brown colour, which should conceal colour changes caused by the addition of iron, which might affect acceptability.
- It has a very strong flavour, and iron salts should not change its taste.

Soy sauce is extracted from the fermentation of soy beans and flour. The literature reports many studies on iron absorption from soy beans and soy products. According to these studies, iron absorption ranges from 2 per cent to 10 per cent (10, 24-26) in human subjects. There are no studies in the literature in which iron absorption from soy sauce has been determined.

Apart from China, soy sauce is also widely used In Japan, Korea, and some countries of Southeast Asia. Iron-fortified soy sauce, therefore, could also be used in these countries. In all, then, about one-third of the population in the world could benefit from the use of iron-fortified soy sauce to prevent iron deficiency.

OBJECTIVES

In this study, we attempted the fortification of soy sauce with ferrous sulphate. First, we studied the solubility of iron and the organoleptic characteristics of the fortified product. We then estimated iron absorption from unfortified and iron-fortified soy sauce. Finally, fortified soy sauce was used In the preparation of vegetable meals in an attempt to determine the influence of bioavailability of some well-known enhancing and inhibiting factors in non-haem iron absorption.

MATERIAL AND METHODS

The soy sauce utilized in all studies was the Seagull brand, produced in Shanghai, PRC. Proximate analysis of this product revealed 65.7 per cent moisture; protein (N x 6.25) 10 per cent; fat 0.1 per cent; crude fibre 0.0 per cent; ash 15.5 per cent, and iron (mg/dl) 15.8 per cent.

a) Chemical analysis

Protein was determined by the Kjeldahl procedure; moisture, crude fibre, ether extract and ash by A.O.A.C. methods. Iron in food-stuffs was determined by a colorimetric technic using bathophenantroline sulphonate, after wet ashing samples with sulphuric acid.

b) Solubility and shelf-life studies:

Soluble iron was measured in samples of soy sauce fortified with ferrous sulphate (Merck Chemical Co.) to final concentrations of 50, 75, 100, 125, 150, and 200 mg of iron per 100 ml. Samples were stored in hermetically sealed glass cylinders at room temperature. Iron was determined in samples from the upper and lower segments of the cylinders after 4, 8, 12, and 16 weeks of storage. The pH of the solutions was measured at 4, 12, and 16 weeks using a Corning 130 pH meter.

After 16 weeks of storage, the unfortified control sample and the products fortified with 100 and 150 mg Fe/dl were tasted by a panel of 13 Chinese judges accustomed to the taste of soy sauce. They were asked to report any differences in taste and any additional tastes they detected.

c) Iron absorption studies:

Absorption studies were performed in 31 healthy volunteer subjects of middle to low socioeconomic status, living in the city of Santiago, Chile, whose ages ranged from 18 to 53 years. Women were over 34 years of age and were engaged in contraception programmes employing lUDs. The studies were authorized by the institutional Committee on Research in Human Subjects and by the Chilean Atomic Energy Commission.

Iron nutritional status was determined before the iron absorption experiments, measuring haemoglobin concentration (cyan-methaemoglobin), haematocrit (microhaematocrit), serum iron and total iron-binding capacity (27), and serum ferritin (28) on a fasting blood sample.

Iron absorption was measured using the extrinsic tag method described by Layrisse and co-workers (12). A total of four absorption tests were performed on each subject in two sets of parallel double isotope measurements. In the first pair of absorption measurements, day 1 and day 2, test meals labeled with different isotopes of iron(59Fe and 55Fe) were administered on successive mornings. Fourteen days following administration of the second test meal, a blood sample was drawn for determination of radioactivity in whole blood. Two additional test meals were then administered on the mornings of the 15th and 16th days of the study, and absorption from these meals was determined from the rise of 55Fe and 55Fe activity in the whole blood obtained 14 days later, the 30th day of the experiment. Calculations of iron absorption were based on the total blood volume estimated from the sex, height, and weight of each subject (29), and a factor of 0.9 was used to correct for the absorbed iron not present in circulating red cells.

The total dose of radioactivity in each test was 4mCi for 55Fe and 1.2mCi for 59Fe for each person. All tests were administered between 9:00 and 10:00 a.m. following an overnight fast. No intake by mouth was allowed for three hours following each test meal. Double isotopic measurements of 55Fe and 59Fe were performed on duplicate 9 to 10 ml samples of whole blood drawn on the 14th and 30th days. These were counted by a liquid scintillation technique using the method of Eakins and Brown (30).

Experiment 1

In this experiment we compared the bioavailability of iron from unfortified soy sauce with that from soy sauce fortified with iron or iron and ascorbic acid. on day 1, each subject received 19 ml of unfortified soy sauce containing 3 mg of elemental iron. On day 2, subjects were given 19 ml of fortified soy sauce containing 23 mg of iron. On day 15, subjects received 19 ml of the fortified sauce plus two moles of ascorbic acid per mole of iron. On day 16, subjects were given a reference dose of iron ascorbate (3 mg iron plus 2 moles ascorbic acid per mole of iron) in 50 ml of deionized water.

Experiment 2

In this experiment vve studied non-haem iron bioavailability from a typical Chinese meal containing iron-fortified soy sauce (75 mg Fe/dl) and the effect of meat and tea on absorption. On day 1, subjects ate the basic meal consisting of 150 g cabbage, 100 g potatoes, 50 g green beans, 100 g rice, 20 g vegetable oil, and 8.5 ml fortified soy sauce labeled with 55Fe. This meal provided a total of about 700 kcal. On day 2, the same meal was given labeled with 59Fe and, immediately after, each individual drank 150 ml of Long Ging green tea prepared with 1.5 g of dry leaves. On day 15, 50 g of rice were substituted for 100 g of ground pork. On day 16, a reference dose of iron ascorbate was given.

Statistical Analysis

Because of the skewed distribution of both iron absorption data and serum ferritin levels, mean values (geometric means) and standard deviations were calculated on the logarithmic scale and the results retransformed as antilog-arithms to recover the original units. Statistical comparisons of iron absorption from any two test meals were performed by Student's t-test applied to the logarithms of absorption tests.

RESULTS

1. Solubility of iron, pH changes, and organoleptic characteristics

Solubility of iron in soy sauce was very good at or below concentrations of 100 mg Fe/dl (Table 1). Concentrations above 125 mg Fe/dl produced precipitation. Solubility changes were noted at four weeks and increased at eight weeks with no further increase. With amounts above 150 mg Fe/dl, precipitation appeared on the third day after the experiment. These results show that iron concentration in ferrous sulphate -fortified soy sauce should not exceed 100 mg Fe/dl.

There were no immediate changes in pH in the fortified products on day 0. With time, pH decreased somewhat in ail samples, including the control. The addition of ascorbic acid had a very modest effect on pH, indicating that soy sauce is a good buffer solution.

Regarding organoleptic characteristics, judges could not find any difference between fortified soy sauce and control, nor could they taste any flavour different from normal.

2. Iron absorption of intrinsic soy sauce iron, ferrous sulphate-fortified soy sauce, and the effect of ascorbic acid on the absorption of iron-fortified soy sauce

The geometric mean iron absorption from unfortified soy sauce (intrinsic soy sauce iron) was 6.95 per cent (Table 2). The absolute amount. absorbed was 0.21 mg (Table 3). The iron absorption from iron-fortified soy sauce was 4.36 per cent, with an absolute amount of absorbed iron of 1.0 mg. Iron absorption from iron. fortified soy sauce with ascorbic acid added increased to 8.35 per cent, the absolute amount of iron absorbed being 1.92 mg. Ascorbic acid almost doubled the absorption of iron-fortified soy sauce, an effect that was highly significant (p<0.001) (Table 2).

FIG. 1. Absolute Amount of Iron Absorbed from Soy Sauce at a Reference Dose Absorption of 40 per cent

FIG.2. Absolute Amount of Iron Absorbed From Soy Sauce Fortified Meals at a Reference

Dose Absorption of 40 Per Cent

TABLE 1. Solubility of Different Amounts of Iron (Ferrous Sulphate) Added to Soy Sauce

Fe Soy Sauce (mg/dl)

Weeks of Observation

  0 4 8 12 16
Control u 14.3 13.5 13.7 14.2 14.0
  L   15.7 13.4 14.7 14.2
50 u 50 4 50.5 49.7 48.5 45.9
  L   57.2 48.2 50.4 54.0
75 u 74 7 68.5 72.7 72.8 64.9
  L   77.2 73.1 73.7 66.2
100 u 106 8 98.8 101 0 83.9 89.5
  L   109.1 98.8 100.6 106.8
100 +A U 105 2 97.4 102.7 100 9 94.8
  L   114.0 98.3 102.5 94.9
125 u 132.2 126.7 102.7 116.2 93.8
  L   152.4 196.4 155.4 141.8
125+A u 1342 116.1 115.1 119.5 111.9
  L   130.1 140.0 131.3 128.4
150 u 138.6 133.9 123.4 128.8 126.0
  L   239.0 233.7 225.6 221.0
200 u 197 3 172.0 174.2 166.4 124.8
  L   332.1 313.0 296.0 315.0

u =upper portion; L =Lower portion; A =Ascorbic acid (Fe:A = 1:2 moles)

TABLE 2. Iron Absorption of Soy Sauce, Fortified Soy Sauce and Fortified Soy Sauce with Ascorbic Acid

Subject Sex Age (yr) Hct. (%) Hgb.(g/dl) Serum
Ferritin
(g/dl)

Iron Absorption (% of Dose)

Absorption Ratio

Soy Sauce
(3 mg Fe)
(A)
Soy Sauce
(23 mg Fe)
(B)
Soy Sauce (23 mg Fe)
+ Asc. Ac
(C)
Reference
Iron
Ascorbate
(D)
A/B
C/A C/B
1 GR F 43 44.1 15.5 15 33.76 29.57 41.68 102.43 1.14 1.23 1.41
2. NC F 34 33.9 10.0 6 46.18 30.83 56.83 98.18 1.50 1.23 1.84
3 AR F 36 42.2 13.7 64 13.24 9.14 18.40 53.94 1.45 1.39 2.01
4. MF F 34 42.4 14.1 59 11.67 6.25 25.06 50.72 1.87 2.15 4.01
5. AB F 40 46.6 16.2 79 8.50 5.94 6.36 42.62 1.43 0.75 1.07
6. SF F 36 40.9 14.2 17 8.28 5.44 8.94 26.78 1.52 1.08 1.64
7. CA F 39 44.0 15.2 28 25.28 9.24 15.90 24.96 2.74 0.63 1.72
8. HG M 43 49.8 16.8 125 8.57 5.82 8.46 23.80 1.47 0.99 1.45
9. RA M 28 52.0 18.1 113 6.09 3.01 5.38 22.06 2.02 0.88 1.79
10.RM M 31 50.2 17.5 88 1.31 1.10 1.66 16.89 1.19 1.27 1.51
11.FV F 36 42.7 13.9 45 7.90 4.48 10.36 16.02 1.76 1.31 2.31
12. FF M 33 51.4 17.3 170 1.26 0.88 2.78 14.46 1.43 2.21 3.16
13. JV M 25 48.4 16.9 155 3.48 1.86 3.00 7.86 1.87 0.86 1.61
14.HS M 34 53.0 18.9 150 0.61 0.41 1.25 4.42 1.49 2.05 3.05
Average   35 45.8 15.6 56 6.95 4.36 8.35 25.73 1.59* 1.20 1.91
S.D.   5 5.3 2.3                

*P<0.001

3. Iron absorption from a vegetable mea/ cooked with iron-fortified soy sauce, and the effect of pork and tea

Table 4 shows the chemically determined nutrient content and the calculated caloric content of the test meals.

The iron absorption from a vegetable meal containing iron-fortified soy sauce was 5.65 per cent; the absolute amount absorbed was 0.50 mg (Tables 5 and 6). Pork enhanced the absorption by 71 per cent, the absolute amount absorbed increasing to 1.08 mg. Green tea inhibited the absorption by 84 per cent; the absolute amount of absorbed iron decreased to 0.27 mg. The effects of tea and meat were highly significant (p<0.001) statistically.

In order to compare the two absorption experiments, we recalculated absorption data, referring them to a reference dose absorption of 40 per cent, as recommended by Hallberg (31) (Tables 5 and 6, Figures 1 and 2). It was found that the percentage of iron absorption from fortified soy sauce and from fortified soy sauce cooked with the vegetable meal was very similar (6.78 per cent and 6.36 per cent, respectively).

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