Technical Paper
Manufacturing Method for Low Salt Dried Fish by Utilizing Used Tea Leaves
2024 Volume 25 Issue 2 Pages 37-43
Details
2024 Volume 25 Issue 2 Pages 37-43
Salt restriction is important in the treatment of hypertension. Therefore, we investigated a method for producing dried fish utilizing used tea leaves that retain most of the components that exhibit antibacterial and antioxidant effects. When treated with a mixture of used tea leaf extract and salt, water content, salt concentration and the number of bacteria were lower than that found for the single substance solution. A synergistic effect was particularly observed for bacterial count reductions when using a 2% salt and used tea leaf mixture. Sensory evaluation showed there was a highly positive evaluation of the dried fish produced with a mixture of 2% salt and used tea leaf extract as compared to commercially available dried fish. Current results suggest that application of used tea leaves during dried fish production can reduce salt while retaining the antibacterial effect, with utilization of used tea leaves thereby expected to reduce food waste.
高血圧の治療には塩分制限が重要である．そこで，抗菌・抗酸化作用を示す成分を多く保持した茶殻を利用した干物の製造方法を検討した．茶殻抽出物と塩の混合物で処理した干物の場合，単独の溶液よりも水分，塩分濃度，細菌数が減少した．とくに，食塩2%と茶殻の混合物を製造した干物の場合，細菌数の減少では相乗効果が観察された．官能評価の結果，食塩2%と茶殻の混合物で製造した干物は，市販の干物に比べて高い評価が得られた．今回の研究結果では，干物製造時に茶殻を使用することで抗菌効果を維持しながら減塩できることが示唆され，茶殻の利用により食品ロスの削減が期待できる．
In Japan, salt restriction is important in the treatment of hypertension, with the Japanese Society of Hypertension recommending less than 6 g per day. The World Health Organization (WHO) has also set a salt reduction target of 5 g for all adults. According to the 2019 National Health and Nutrition Survey of the Ministry of Health, Labour and Welfare, the average salt intake is 10.1 g, at 10.9 g for men and 9.3 g for women. Therefore, it is important to refrain from salt intake on a daily basis and to devise cooking methods that reduce the salt content of food. Recently, there have been reports of low-sodium bread that uses the salty taste-enhancing effect of soy sauce and low-salt bread that uses the salty taste-enhancing effect of soy sauce. [1-3]
When using salt during the preparation of dried fish, it produces antibacterial action, protein coagulation action, reduction of water activity and an influence on the taste. Processed dried fish is generally salted in two ways: dry salting and brine salting. In the past, the salt water concentrations were maintained at 15 - 25%, with adjustments made according to the freshness, lipid content, weather, etc., while the soaking time was about 3 to 4 hours, depending on the size of the fish. Recently, the cubic salt method has become the mainstream method for salting dried fish. When using this method, the fish is allowed to soak in 10-20% salt brine for 15-20 minutes [4]. Drying methods include sun drying, hot air drying, and cold air drying. Sun-dried fish has a good texture and flavor, and in addition, can be used to make traditional dried fish. However, since fish cannot be dried on a rainy day, this has the disadvantage of being affected by the weather. Therefore, hot air drying and cold air drying are now the mainstream methods being used. Hot air drying is a method of drying fish at a temperature of 30°C or higher for 30 to 100 minutes [4, 5]. The most serious problem in preserving food, including dried fish, is deterioration that occurs due to the proliferation of microorganisms. However, due to the current health consciousness trends, there has been an effort to try and reduce salt intake, and as a result, the control of microorganisms in the context of reduced salt usage has now become an important issue.
Green tea is known to contain many ingredients that have been shown to have a positive effect on health. Catechins, which are the most abundant of the substances found in green tea, have been reported to have antioxidant [6] and anti-cancer [7,8] effects as well as antibacterial [9-12] anti-arteriosclerotic, anti-viral and anti-allergic [5]. Caffeine has been shown to be a central nervous system stimulant, prevent drowsiness, and have cardiotonic and diuretic effects. Gamma-aminobutyric acid and theanine have blood pressure lowering and anti-stress effects [13-15]. When making green tea for drinking, most of the active ingredients, such as catechin, caffeine, and tannin, which are contained in the green tea leaves, are not extracted. In the green tea leachate composition data that are listed in the Standard Tables of Food Composition in Japan, only about 20 to 30% of the active ingredients of green tea are eluted [16-18]. Therefore, used green tea leaves that are considered to be food waste still have many active ingredients that remain after the original use of the leaves. Currently, tea leaf waste is reused in fertilizer for plants, in bath salts, in deodorizers, and in treatments for athlete’s foot, all of which are designed to have antibacterial effects [19].
In order to reduce salt intake and reduce food loss, we investigated a new dried fish production method based on the antibacterial effect of green tea leaves. However, used green tea leaves are not normally reused as food. Moreover, in Japan, it is essential to reduce food waste, including used tea leaves, or in other words, reduce food loss. Thus, we investigated a new dried fish production method using the antibacterial effect of green tea leaves in order to help reduce salt intake in addition to reducing food loss.
Green tea was made from tea（ordinary Sencha）grown in Suzuka City. Yeast extract were obtained from Nakarai (Kyoto, Japan). Glucose, hipolypepton and sodium chloride (NaCl) were obtained from Fujifilm Wako Pure Chemical Corporation (Osaka, Japan), while sterile petri dishes were purchased from As One Corporation (Osaka, Japan). The electronic moisture meter used was the MOC63 device obtained from Shimadzu Corporation (Kyoto, Japan). The digital salinity meter was obtained from PAL-SALT of ATAGO Corporation (Tokyo, Japan). Measurement accuracy is ±0.05% (0.00 to 0.99%) of indicated value and ±5% (1.00 to 10.0%) relative accuracy. The homogenizer (Stomacher) and sterilization filter bags were obtained from Pro Media/ SH M of ELMEX (Tokyo, Japan).
2.2 Each methods 2.2.1 Green tea extraction methodUsing the method listed in the Japanese Food Standard Composition Table, green tea and hot water (90°C) were heated at a ratio of 1:40 (w/w) for 1 minute, and then the solution and used green tea were separated. Furthermore, used sencha and hot water (90˚C) were heated at a ratio of 1:2 (w/w) for 10 minutes to form a solution and recovered as a wet used sencha extract. Used sencha extract contains many active ingredients such as catechins and amino acids.
2.2.2 Dried product processing methodThe fish used were horse mackerel (Trachurus japonicus) caught in Ise Bay, which were purchased at a market in Suzuka City, Mie Prefecture, weighing around 200 g and having a body length of 25 to 30 cm. The fish were used for the experiment within one day. As a sample, 100 horse mackerel were used. Removed the head, internal organs, bones and skin of the horse mackerel, washed it with water, three fillets grated, and prepared two pieces of meat (approximately 50g × 2).
2.2.3 Dried fish manufacturing methodDried fish manufacturing method was as follows: each sample (n = 10) was placed in a container, with the sample then completely immersed in either water, a salt brine (final salt concentrations were 16%, 8%, 4%, 2%, 0.4% or 0.2%), used green tea leaf extract, or a mixture (1:2(v/v) of used green tea leaf extract and a salt brine (final salt concentrations were 16%, 8%, 4%, 2%, 0.4% or 0.2%), with an immersion time of 5 hours and room temperature. After the immersion, the samples were rinsed once in water and then drained using kitchen paper towels. In the drying method, samples were dried overnight at room temperature, using the traditional method.
2.3 Measurements 2.3.1 Measurement of moisture contentTo measure the water content, 2 g of the sample was heated and dried at 200°C using an electronic moisture meter. The measured value was determined at the point where the rate of change of the water content for 30 seconds was 0.05% or less.
2.3.2 Measurement of SalinityThe salt concentration was measured by directly inserting a Pal-Salt Probe into three different points on the sample. This value is calculated as g per 100 g.
2.3.3 Measurement of bacterial countsBacterial counts were measured using the plate pour method [20]. The methods were done by adding 9 mL of phosphate buffer (pH7.4) to 1 g of the sample followed by homogenization with a homogenizer for 1 minute in order to prepare a crude extract. The crude extract was diluted with phosphate buffer (pH7.4), with 1 mL diluted in 9 mL of the medium followed by mixing, transfer to a culture plate（standard agar medium）and then cultured at 37°C for 24 hours. After culturing, bacteria numbers were then counted. The composition of the standard agar medium was prepared by adding 2.5 g of yeast extract, 5 g of hipolypeptone, 1.5 g of glucose, and 15 g of agar to 1 liter of water (pH7.4).
2.4 Sensory evaluation of the manufactured dried fishThis study was conducted with the approval of the Suzuka University of Medical Science Clinical Research Review Committee (approval number 485). Sensory evaluation was performed based on JIS Z 9080:2004 (ISO 8588, 85891). The materials used for the sensory evaluation included dried horse mackerel purchased at a market in Suzuka City, and dried horse mackerel samples prepared using water, 2% salt concentration, used green tea leaf extract, and mixed solutions. Subjects taking part in the study included 20 men and women in their 20s. The heat dried fish on a gas stove before eating. We placed the following samples on a dish that was labeled ABCD, in which A was the dried horse mackerel made with water, B was the dried horse mackerel made with 2% salt concentration, C was the dried horse mackerel made with used green tea leaf extract, and D was dried horse mackerel made with a mixture of 2% salt concentration and used green tea leaf extract. The standard purchased dried horse mackerel was positioned on the dish so as to alternate with the sample, with evaluations done in order starting from A. The dried horse mackerel purchased from the market was used as a standard sample and used to compare with each of the produced dried horse mackerel samples. The samples were analyzed for 6 items: appearance, texture, hardness, odor, taste, and comprehensive evaluation. The evaluation was a semantic differential method that produces evaluations based on a five-level evaluation scale of “good (＋2), somewhat good (＋1), normal (0), somewhat bad (-1), and bad (-2)”. After performing the evaluations, the evaluation contents were averaged and the results reviewed. The evaluation of the standard sample shall be normal (0).
2.5 Statistical analysisStatistical software IBM SPSS Statistics 26 (IBM Japan, Tokyo) was used for statistical analysis. Multiple group data were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparison test. Comparisons between two groups were performed with an unpaired two-tailed Student’s t-test. All values are expressed as means ± SD and were considered significant when p-values were less than 0.05.
We measured the salinity and moisture content (%) of dried horse mackerel sold commercially, and found that the salinity ranged from 1.4 to 1.8% and the moisture content was about 60%. The Japanese Food Dictionary describes the processing method for dried fish as 10 to 20% salt concentration and 4 to 5 hours of soaking time for soaking conditions [19]. Therefore, we examined processing methods that satisfied these conditions. Although detailed analysis data are not shown, we were able to obtain almost the same salt concentration and moisture content (%) of commercially available dried fish by using a salt concentration of 16% for soaking, soaking time of 5 hours, and drying overnight. We have previously reported that used green tea leaf extract suppresses histamine-producing bacteria with the extract having no inhibitory effect unless the concentration was high [21]. During the processing and production of dried horse mackerel, the used green tea leaf extract had an antibacterial effect with the remaining active ingredient used as a dipping solution, after which the water content, salt content and bacterial count were measured. The dried horse mackerel treated with the used green tea leaf extract had almost the same water content and salt concentration as that found for the samples treated with water. There was much weaker suppression of the number of bacteria in the dried horse mackerel treated with used green tea leaf extract as compared to the dried horse mackerel that was treated with 16% salt brine. However, the bacterial number was suppressed as compared to that observed for the dried horse mackerel that was treated with water (Fig. 1). Therefore, these results showed that the dried horse mackerel produced using the green tea leaf extract inhibited the growth of bacteria in dried fish in the same manner as saline.
Moisture (A), Salt content (B) and Bacterial count (CFU/g) (C) of dried fish produced by each method. Data are presented as the mean ± SD of five experiments; H2O versus 16% NaCl and used tea leaf extracts. Significant difference, **p < 0.01, *p < 0.05.
Subsequently, in order to investigate the inhibitory effect of adding salt water to the green tea leaf extract that was used in the salt reduction examination, we investigated the use of different final salt concentration mixtures (16%, 8%, 4%, 2%, 0.4% and 0.2%) to produce dried horse mackerel using green tea leaf extract, with moisture content, salt content, and bacteria count then analyzed. There was little change observed for the moisture content between the dried horse mackerel samples produced with water and the dried horse mackerel samples produced with the different concentrations of the salt brine. Comparisons of the dried horse mackerel samples made with water versus those made with the mixed liquid showed that the moisture content of the samples produced with the final salt concentration mixtures of 16%, 8%, 4% and 2% tended to be lower. However, the water content of the samples produced using the final salt concentration mixtures of 0.4% and 0.2% were similar to those for the samples produced with water, salt brine, and the used green tea leaf extract (Fig. 2). Comparing the dried horse mackerel samples prepared at final salt concentrations of 16%, 8%, 4%, and 2% with samples prepared at the same salt concentrations by mixing spent green tea leaf extract, the salinity of the samples adjusted with the mixture tended to be lower. (Fig. 3). The number of bacteria found in the dried horse mackerel samples prepared at each salt concentration was lower than that observed for the samples prepared with water. In particular, the number of bacteria was low in the sample of dried horse mackerel prepared at a high concentration. Furthermore, at final salt concentrations of 16% to 4%, 0.4% and 0.2%, there was no further suppression of the bacterial count observed after the addition of the used green tea leaf extract. However, at the final salt concentration of 2%, there was a slight suppression of the number of bacteria in the dried horse mackerel samples that were prepared by adding used green tea leaf extract (Fig. 4). Therefore, these results suggest that the production of dried horse mackerel samples when using a 2% salt concentration combined with the used green tea leaf extract can synergistically suppress the growth of bacteria. It is thought that the synergistic inhibition of bacteria by the mixture of salt and used green tea leaf extract may be due to the antibacterial action of catechins.
Moisture of dried fish prepared using the different salt concentrations, used green tea leaf extract, and the mixed solution of each salt concentration with the used green tea leaf extract. Data are presented as the mean ± SD of five experiments; H2O versus 16% NaCl, 8% NaCl, 4% NaCl, 2% NaCl, 0.4% NaCl, 0.2% NaCl, used tea leaf extracts, 16% NaCl ＋ used tea leaf extracts, 8% NaCl ＋ used tea leaf extracts, 4% NaCl ＋ used tea leaf extracts, 2% NaCl ＋ used tea leaf extracts, 0.4% NaCl ＋ used tea leaf extracts, and 0.2% NaCl ＋ used tea leaf extracts. Significant difference, *p < 0.05. Each salt concentration versus each salt concentration ＋ used tea leaves extracts. Significant difference, p < 0.05.
Salt content of dried fish prepared using the different salt concentrations, used green tea leaf extract, and the mixed solution of each salt concentration with the used green tea leaf extract. Data are presented as the mean ± SD of five experiments; H2O versus 16% NaCl, 8% NaCl, 4% NaCl, 2% NaCl, 0.4% NaCl, 0.2% NaCl, used tea leaf extracts, 16% NaCl ＋ used tea leaf extracts, 8% NaCl ＋ used tea leaf extracts, 4% NaCl ＋ used tea leaf extracts, 2% NaCl ＋ used tea leaf extracts, 0.4% NaCl ＋ used tea leaf extracts, and 0.2% NaCl ＋ used tea leaf extracts. Significant difference, **p < 0.01, *p < 0.05. Each salt concentration versus each salt concentration + used tea leaves extracts. Significant difference, p < 0.01.
Bacterial count of dried fish prepared using the different salt concentrations, used green tea leaf extract, and the mixed solution of each salt concentration with the used green tea leaf extract Data are presented as the mean ± SD of five experiments; H2O versus 16% NaCl, 8% NaCl, 4% NaCl, 2% NaCl, 0.4% NaCl, 0.2% NaCl, used tea leaf extracts, 16% NaCl ＋ used tea leaf extracts, 8% NaCl ＋ used tea leaf extracts, 4% NaCl ＋ used tea leaf extracts, 2% NaCl ＋ used tea leaf extracts, 0.4% NaCl ＋ used tea leaf extracts, and 0.2% NaCl ＋ used tea leaf extracts. Significant difference, **p < 0.01. Each salt concentration versus each salt concentration ＋ used tea leaves extracts. Significant difference, p < 0.05.
The moisture content of dried fish produced by drying at a constant temperature (30°C) has been shown to be lower than the moisture content of dried fish produced by drying in the sun for a long time. This is likely due to the fact that during the former process the water evaporates both due to being dried under the sun during the day and its exposure to air overnight. Even so, sun-dried fish can be easily affected by the weather, temperature, and wind strength, and thus, this method might not be suitable for consistent production of dried fish. In this case, it is dried at room temperature, and evaporation of water occurs during the process. However, when a used green tea leaf extract is used, it is thought that the oxidation of lipids is suppressed by the antioxidant effect of tea. After salting, there is a change in the water content and weight of the fish after the salt penetrates into the it [22]. Salt permeation of fish is determined by the amount of salt used, and the permeation rate, with the amount of permeation increasing as the amount of salt used increases. In our current analysis, we found that there were no significant changes in the water content whether dried fish was made using a salt brine or using green tea leaves. However, there was a decrease in the water content of the dried fish prepared from a mixed solution of the saline and the used green tea leaf extract. This is considered to be a synergistic effect that occurs when mixing these two solutions. The salinity of the dried product that was prepared with the mixed solution of the salt brine and the used green tea leaf extract was lower than that found for the salt concentration of the dried product that was prepared with only the salt brine. These findings demonstrate that the used green tea leaf extract contains components that are able to reduce both water content and salinity. The synergistic effect on reducing water and salt content by mixing salt and used green tea leaf may be due to the osmotic pressure effect of minerals such as potassium.
While bacteria numbers have been found to range from 2.9 × 103 to 3.0 × 104 / g in the epidermis of commercially available horse mackerel and 1.5 × 103 to 1.9 × 103 / g in the muscle, these bacteria can vary depending on the place and time of collection [23]. In the present study, we measured the total number of bacteria in the epidermis and muscle. In the dried fish that was prepared with the used green tea leaf extract, analysis showed that there was suppression of the growth of the bacterial count. However, no inhibitory effect was observed for dried fish that was prepared using a 16% salt brine. The main component of the bactericidal action of tea is catechins, with this component known to have an antibacterial action (bactericidal action, bacterial exotoxin inhibitory action, bacterial enzyme inhibitory action, etc.) and an antiviral action [13]. Among the catechins present in tea leaves, epigallocatechin gallate (EGCG), epicatechin gallate (ECG), and epigallocatechin (EGC) show bactericidal action, with the strength of action in the order of ECG> EGCG> EGC. However, it has also been reported that epicatechin (EC) and catechin (C) have almost no bactericidal action [24]. The bactericidal action of these catechins strongly changes the galloyl group and pyrogallol group. Theaflavin digallate (TF3), which has two galloyl groups and is formed by the oxidative condensation of EGCG and ECG, has the strongest bactericidal action. In our results as well, catechins were considered to be involved in the antibacterial action.
We performed a sensory evaluation of four types of dried horse mackerel samples, which included our prepared samples along with commercial samples (Fig. 5). The moisture content of the commercially available dried fish used this time was 64.1%, and the salt content was 1.6%. As for the appearance, the four kinds of prepared horse mackerel samples had a very low evaluation as compared with the commercial dried horse mackerel samples. There was no difference in terms of texture and hardness between the commercially dried horse mackerel samples and the samples prepared utilizing the used green tea leaf extract. However, samples prepared from a mixture of salt and used green tea leaf extract were given a high sensory evaluation as compared to the commercially dried horse mackerel samples. In terms of taste, the dried horse mackerel samples that were produced by mixing salt and used green tea leaf extract also were given a high sensory evaluation as compared with the commercially dried horse mackerel samples. During the comprehensive evaluation, the dried horse mackerel samples prepared using a mixed solution of salt and the used green tea leaf extract were also given a high sensory evaluation as compared with the commercially dried horse mackerel samples. Thus, overall, although the dried horse mackerel samples prepared using a mixed solution of salt and used tea leaf extract had a poor evaluation with regard to the appearance, they were highly evaluated with regard to the texture, hardness, and taste as compared with the commercially available dried horse mackerel samples. During the sensory evaluation (data not shown), results of the observation of the appearance of the fish after being soaked and dried indicated that the dried fish prepared by soaking in 2% salt brine did not have as much gloss and tightness. In contrast, the dried fish prepared by immersing the fish in the used green tea leaf extract picked up the color of the green tea leaves. As a result, subjects found the appearance of the dried fish produced by immersing the fish in the used green tea leaf extract was worse than that of the commercially available dried fish. However, the dried fish prepared with a mixed solution of used green tea leaf extract and 2% salt obtained a good evaluation including for the comprehensive evaluation. The reason for this is believed to be due to the synergistic effect of the low concentration of salt and the compounds contained in the green tea leaves. Tea leaves contain polyphenols, amino acids, caffeine, sugars and organic acids. Of these, the balance between the umami and sweetness of amino acids and the bitterness and astringency of polyphenols is important for the taste of green tea. Among the many free amino acids contained in tea leaves, theanine is thought to play a major role in the unique taste of tea. Theanine becomes bitter at high concentrations. Since the theanine concentration in the exudate was as low as the threshold value (0.1-0.6%), it was believed that the tea provided sweetness and umami rather than bitterness [25]. It has been reported that theanine has a synergistic effect on umami and at the same time suppresses acidity. Therefore, it can be expected to be used as an umami seasoning, similar to glutamic acid, or as a flavor improving agent. Furthermore, the results of this research are thought to lead to the reduction of food waste.
Sensory evaluation of manufactured dried fish. A total of 20 men and women in their 20s evaluated and analyzed the contents for 6 items: appearance, texture, hardness, odor, taste, and comprehensive evaluation. H2O; Open column, used tea leaf extracts; light gray column, 2% NaCl; dark gray column, 2% NaCl ＋ used tea leaf extracts; black column. The evaluation of the standard sample shall be normal (0).
The results of this study are: 1) Using tea when producing dried fish shows a salt-reducing effect, 2) People with high blood pressure can consume dried fish without worrying about salt content, and 3) Since used tea leaves are used as food waste, food loss can be eliminated. These results suggest that the results have implications for both the environment and humans. In the future, we would like to conduct sensory evaluations for a wider age group and expand the social reach of dried fish made from used tea leaves based on scientific evidence.
The authors would like to thank Aya Kotou, Mi Ukita, Risa Hatanaka, Anaho Sasaki, Yki Maeda, Izuki Suzuki and Yuna Hirano for technical assistance with the experiments.
