Production method for tomatine-containing extraction liquid and treatment method for tomato residua

Abstract

The present invention makes it possible to easily produce tomatine and obtain tomato residua at low cost from discarded tomato residua. This production method for a tomatine-containing extraction liquid uses the portion of tomato plants that is not the fruit as starting tomato residua and includes a step for estimating the water content and the dry weight of the starting tomato residua and a step for immersing the starting tomato residua in a starting extraction liquid that has a ratio of ethanol and acetic acid of 380:1 and has been prepared such that the abovementioned water content is 4%–6% of the whole. Repeating the steps makes it possible to increase the tomatine concentration of a tomatine-containing extraction liquid, and the water of the tomato residua is replaced with ethanol during the extraction, making it possible to obtain tomato residua by leaving to dry for about 24 hours.

Description

Method for producing tomatine-containing extract and method for treating tomato residue

[0001] The present invention relates to a method for producing tomatine from tomato residues such as stems, leaves, and roots after harvesting tomato fruits, and a method for treating tomato residues.

[0002] Tomato residues, such as leaves and stems other than the fruit after harvesting, are usually disposed of as industrial waste. Dutch-style hydroponic tomato cultivation, which has been attracting attention in recent years, is a highly efficient farming method, but it also produces large amounts of residues other than the fruit, such as leaves and stems. Because this tomato residue contains approximately 90% water, burning it requires high fuel costs. Therefore, there was a need for an effective use of tomato residues.

[0003] Meanwhile, tomatine, a toxic component found in tomatoes, has been attracting attention as an effective use of tomato residue. Tomatine is a toxic component found in tomatoes that is not toxic to humans, but is used as an insect repellent to protect tomatoes from insects.

[0004] Patent Document 1 discloses a method for producing tomatine using tomato residue. According to Patent Document 1, the tomato residue is first dried. This is because tomato residue contains a large amount of moisture, and drying it in this state is necessary to improve workability. The preferred drying conditions for the tomato residue are drying at 50 to 70°C for 5 to 20 hours to reduce the moisture content to 5% or less. Patent Document 1 also discloses extracting tomatine from the dried tomato residue using methanol or the like.

[0005] Patent Document 2 discloses a method for extracting the glycoalkaloid tomatine by adding 3 to 15 times by weight of water to tomato residue that has been cut or dried and then cut, and then adding an acid solution to adjust the pH to 2 to 5, followed by stirring and mixing at a temperature between room temperature and 80°C for 30 to 120 minutes.

[0006] Patent Document 3 points out the problem that, when Patent Document 2 was actually tried, tomatine could only be extracted from parts naturally rich in tomatine, such as axillary buds and leaves, and that when conventional methods were applied to green tomatoes, only small amounts of tomatidine were obtained, with much of the tomatine remaining. Patent Document 3 then goes on to disclose a method for extracting tomatidine from green tomatoes with a higher yield than conventional methods. The method discloses a step of mixing juice and / or a water extract from green tomatoes with ethanol, removing the precipitate, and obtaining a supernatant fraction containing tomatine.

[0007] Japanese Patent Laid-Open No. 9-020608 Japanese Patent Laid-Open No. 2018-184394 Japanese Patent Laid-Open No. 2020-203856

[0008] The methods of Patent Documents 1 and 2 have the problem that steps such as drying the tomato residue and subsequent concentration are required to obtain tomatine from the tomato residue. Furthermore, since a large amount of water remains in the tomato residue after tomatine extraction, the tomato residue still contains water, and thus the method has to be disposed of as industrial waste in the same manner as in the past, which has not yet been solved.

[0009] The present invention has been conceived to solve the above-mentioned problems, and provides a method for producing a tomatine-containing extract which does not require drying the tomato residue when extracting tomatine from the tomato residue, and which is easy to do and reduces the labor required for concentration.

[0010] The present invention also provides a method for treating tomato residues, which can omit a drying step when treating the tomato residues after tomatine extraction as industrial waste.

[0011] More specifically, the method for producing a tomatine-containing extract according to the present invention is characterized by comprising the steps of: estimating the water content and dry weight of original tomato residue, which is the parts of the tomato plant other than the fruit; and immersing the original tomato residue in an original extract having an ethanol:acetic acid ratio of 380:1, which has been prepared so that the water content is 4% to 6% of the total.

[0012] Furthermore, a method for treating tomato residue according to the present invention is characterized in that it comprises the steps of: estimating the moisture content and dry weight of the original tomato residue, which is the portion of the tomato plant other than the fruit; immersing the original tomato residue in an original extract solution having an ethanol to acetic acid ratio of 380:1, which has been prepared so that the moisture content is 4% to 6% of the total; and removing the original tomato residue.

[0013] In the method for producing a tomatine-containing extract according to the present invention, the amount of water contained in tomato residue is calculated as part of the extract, and the tomatine is extracted from the tomato residue together with the water by immersing the tomato residue in a mixed solution of ethanol and acetic acid at a predetermined ratio, thereby enabling efficient extraction of tomatine from the tomato residue.

[0014] Furthermore, by adding to the extract an amount of ethanol equal to the amount of liquid carried over by the tomato residue separated from the extract, multiple extractions can be performed at the same extraction rate from the second time onwards. This multiple extractions allows the tomatine concentration in the extract to be concentrated in proportion to the number of extractions, which has the effect of reducing the effort required for concentration.

[0015] Furthermore, in the tomato residue treatment method of the present invention, the water content in the residue is replaced with a mixture of ethanol and acetic acid, so there is no need to dry the tomato residue before treatment. The tomato residue removed from the extract can be left overnight to evaporate the ethanol, allowing it to be treated as industrial waste with a low water content.

[0016] 1 is a schematic diagram illustrating the steps of the method according to the present invention. 2 is a graph showing the relationship between the number of repetitions and the tomatine content per 4 L of extract.

[0017] The method for producing a tomatine-containing extract and the method for treating tomato residue according to the present invention will be described below with reference to examples and drawings. Note that the following description exemplifies one embodiment of the present invention and one example, and the present invention is not limited to the following description. The following description can be modified within the scope of the present invention.

[0018] Furthermore, embodiments and examples obtained by appropriately combining the technical means disclosed in different embodiments and examples are also included in the technical scope of the present invention. Furthermore, all documents described in this specification are incorporated herein by reference. In this specification, when a numerical range is described as "A to B," the description means "A or more (greater than A, including A) or B or less (smaller than B, including B)."

[0019] The tomatoes that can be used as ingredients in the present invention are not particularly limited as long as they are tomatoes. Medium to small tomatoes (cherry tomatoes), which are often fully ripened on the tree, are suitable for use. Also suitable for use are tomatoes that can be harvested in bunches. Examples of tomatoes that can be harvested in bunches include Kosuzu, Carol 7, Niagara Sweet, Rouge de Bordeaux, Genary, and Redley.

[0020] Tomato residue is the leaves and stems of tomatoes, but may also include offshoots and roots. Fruit parts are not included.

[0021] Figure 1 shows an outline of the steps of the method for producing a tomatine-containing extract and the method for treating tomato residue according to the present invention. The method for producing a tomatine-containing extract and the method for treating tomato residue according to the present invention are composed of the same steps, but are classified as a method for producing a tomatine-containing extract or a method for treating tomato residue depending on what is extracted. Therefore, hereinafter, these methods are collectively referred to as "the method according to the present invention."

[0022] The method according to the present invention is divided into an initial process and subsequent processes. Alternatively, only the initial process may be performed. In FIG. 1, the initial processes are represented by (a) to (d), and are collectively referred to as process [I]. The subsequent processes are represented by (e) to (i), and are collectively referred to as process [II].

[0023] The method according to the present invention includes a step of estimating the moisture content and dry weight of tomato residue. The method for measuring the moisture content Win of tomato residue 1 is not particularly limited. The moisture content Win of tomato residue 1 may be measured for each unit to be treated, or a calibration curve of the moisture content per unit weight as a function of the time elapsed since the tomato residue 1 was produced may be obtained in advance, and the moisture content Win may be estimated based on the time elapsed since the tomato residue 1 was produced and the weight A to be treated. The dry weight DA of tomato residue 1 may be calculated by subtracting the moisture content Win from the weight A of tomato residue 1.

[0024] The time when tomato residue is produced refers to the time when tomato fruits are harvested and the stems and leaves are separated from the roots or when the roots are removed from the medium (including soil). Tomato residue 1 in Figure 1(a) is also called original tomato residue.

[0025] Next, original extract 2 is prepared as the extract for process [I]. The original extract for process [I] may also be referred to as initial extract 2. The original extract is a solution in which the moisture content of the residue is 5% of the total, and ethanol and acetic acid are mixed at a weight ratio of 380:1. At this time, the acetic acid becomes a 5% solution. In other words, the water constituting the 5% acetic acid includes the moisture content Win of tomato residue 1. In Figure 1, when ethanol is We (g), acetic acid is Wac (g), and water is W (g), the amount of water is the sum of the moisture content Win (g) and the amount of water added as water Wout (g). Note that ethanol, acetic acid, and water may be converted by volume assuming a specific gravity of 1.

[0026] Figure 1(b) shows the state of original tomato residue 1 immersed in original extract solution 2. At this point, the water content (W in) of original tomato residue 1 is 5% of the total, and the original extract solution 2 is a mixture of ethanol and acetic acid at a weight ratio of 380:1. The acetic acid concentration is 5%. Therefore, the solution prepared in Figure 1(a) is solution 2p, which is a mixture of ethanol (W g), acetic acid (W g), and water (W out g). This may also be referred to as initial extract precursor solution 2p. Therefore, original tomato residue 1 is immersed in original extract solution 2, which has a water content of 5% of the total and an ethanol and acetic acid ratio of 380:1 (Figure 1(b)). This immersion is carried out at room temperature for 72 hours. Immersion may be performed with agitation in the dark.

[0027] This soaking step increases the tomatine concentration in original extract 2, and as will be described later, the water content Win in original tomato residue 1 is almost eliminated. The reason for this is not clear, but because water and ethanol have high affinity and diffuse uniformly into each other, the ethanol in original extract 2, which contains a high concentration of ethanol, replaces the water in original tomato residue 1, and it appears that tomatine is extracted into original extract 2 together with the water. Original extract 2 from which tomatine has been extracted is a tomatine-containing extract. It is sufficient for the method for producing a tomatine-containing extract in process [I] to include the steps up to this point.

[0028] After the soaking step, the original tomato residue 3 is pulled up. The pulled-up original tomato residue is replaced with the symbol 3 because the water content Win has been replaced with ethanol. It is sufficient for the tomato residue processing method in process [I] to include the steps up to this point.

[0029] The amount of liquid removed by the original tomato residue 3 from the original extract 2 is then examined. If the amount of liquid removed is denoted as Cout (g), then this can be calculated by subtracting the dry weight DA (g) from the weight A' of the original tomato residue 3. The weight of the original tomato residue 3 is represented by the symbol A' in relation to the weight A of the tomato residue 1. Furthermore, while the dry weight DA (g) is an estimated value, the actual measured weight of the tomato residue 4 obtained by completely drying the original tomato residue 3 (described below) can be taken as DA', and the amount of liquid removed Cout (g) can be calculated as A - DA' (A minus DA'). It takes time to dry the weight DA' of the tomato residue 4, but evaporation can be prevented by sealing the extract during this time.

[0030] Furthermore, as will be described later, the volume of original liquid extract 2 obtained by separating original tomato residue 3 can be measured, and the amount of liquid carried away by original tomato residue 3 can be determined by measuring the volume of original liquid extract 2 relative to the volume of original liquid extract 2 in the state shown in FIG. 1( b).

[0031] In the original tomato residue 3 pulled up from the original extract 2, most of the water contained therein has been replaced with ethanol. Therefore, if left for about 24 hours, the ethanol evaporates, and tomato residue 4 ( FIG. 1( d) ) with a low water content can be obtained. Furthermore, the original tomato residue 3 pulled up from the original extract 2 may be subjected to a drying process.

[0032] The drying step is not particularly limited, and drying can be performed by simply leaving it alone, exposing it to air at room temperature (or room temperature) (air drying), leaving it in a high-temperature environment, or exposing it to warm air in a high-temperature environment. Physical methods such as a centrifugal evaporator can also be used. Whichever method is used, the amount of water contained in the original tomato residue 3 is small, and most of it has been replaced by ethanol, so drying can be performed in less time (and at less cost) than when the water content has not been replaced by ethanol.

[0033] Through the above series of steps, the original extract 2 becomes a tomatine-containing extract, and the original tomato residue 3 becomes a tomatine residue 4 from which the water content has been removed, which can be disposed of at low cost as industrial waste that does not contain water.

[0034] Next, we will explain process [II]. In original extract 2 in Figure 1(c), the liquid equivalent to Cout (g) carried over from original tomato residue 3 during separation is gone. Considering the state of tomato residue 4, this is thought to be almost entirely ethanol. Therefore, in process [II], first, the amount of ethanol carried over is added to original extract 2 (Figure 1(e)), and this is used as a new extract (new extract 6). Then, the same amount (A (g)) of tomato residue as original tomato residue 1 is immersed in new extract 6 as new tomato residue 7 (Figure 1(f)). Note that because the extract is a highly concentrated ethanol, it is not prohibited to anticipate the amount of evaporation that occurs in each step and add more ethanol to the amount of ethanol carried over.

[0035] 1 shows that the amount of liquid carried out is calculated in weight terms, but it is also possible to measure the volume of original extract 2 after original tomato residue 3 has been separated off, and add ethanol to the original extract 2 so that the volume becomes the same as that of solution 2p. In other words, the amount of liquid carried out by original tomato residue 3 may be calculated by subtracting the volume of original extract 2a after original tomato residue 3 has been separated off from the volume of original extract 2 immediately after original tomato residue 1 has been immersed.

[0036] The moisture content Win is also estimated for new tomato residue 7. If the weight is the same as that of original tomato residue 1, the moisture content Win can be considered to be approximately the same. Therefore, if the weight of new tomato residue 7 is measured, the moisture content Win can be considered to have been estimated.

[0037] Because the moisture Win contained in the original tomato residue 1 has already been transferred to the new extract 6, when new tomato residue 7 is added, the amount of moisture in the new extract 6 exceeds the amount of moisture in the initial extract 2 of process [I], as shown in Figure 1(g). However, in the method of the present invention, it has been confirmed that the amount of tomatine extracted does not change up to five times after repeating this process, even if the moisture content increases.

[0038] The new tomato residue 7 is immersed in the new extract liquid 6 ( Figure 1 (g)). The immersion is the same as that in Figure 1 (b), and is carried out at room temperature for 72 hours. During this immersion, the residue may be kept in a light-shielded environment and stirred. Once the immersion process is complete, the original tomato residue 8 is removed ( Figure 1 (h)). Because the water contained in the separated new tomato residue has been replaced with ethanol, the new extract liquid and the separated new tomato residue are referred to as the original extract liquid 6a and the original tomato residue 8. At this time, the amount of liquid (Cout (g)) that the original tomato residue 8 has removed from the original extract liquid is measured. By leaving this original tomato residue 8 for 24 hours, the ethanol will evaporate, and a tomato residue 9 with a low water content can be obtained.

[0039] Through the above series of steps, the original extract 6a becomes a tomatine-containing extract having an increased tomatine concentration, and the original tomato residue 8 has its water content removed, allowing it to be disposed of as industrial waste at low cost.

[0040] In the method according to the present invention, the step of process [II] can be repeated multiple times. That is, the original extract 6a and original tomato residue 8 obtained in process [II] are replaced with original extract 2a and original tomato residue 3, respectively, and the step of process [II] is repeated. More specifically, this replacement can be performed after the completion of the immersion step in Figure 1(g). In this case, the amount of ethanol added in Figure 1(e) is the amount of liquid removed (Cout (g)) measured in Figure 1(h).

[0041] Example 1: Comparison of Extraction Amounts The amount of tomatine extracted was compared between a case where the original tomato residue was dried (Comparative Example) and a case where it was not dried (Example). First, for the Comparative Example, 100 g of tomato stems and leaves were prepared and dried at 60°C for 24 hours. Tomato stems and leaves contain approximately 90% water, and this drying procedure reduced the amount of tomato stems and leaves to approximately 10 g.

[0042] A mixture of 1900 mL of ethanol, 5 mL of acetic acid, and 95 mL of water was prepared and used as Extraction Solution B. The dried stems and leaves were each immersed in 200 mL of Extraction Solution B and stirred at room temperature in a dark environment for 72 hours. The case of the stems is referred to as Comparative Example (Stem), and the case of the leaves is referred to as Comparative Example (Leaf).

[0043] Meanwhile, as an example, 100 g of tomato stems and leaves were prepared. Approximately 90% of this 100 g contained water. Extract A was prepared by mixing 1900 mL of ethanol, 5 mL of acetic acid, and 5 mL of water. The water content was 90 mL less than that of Extract B, but this missing water was contained in the 100 g of stems and leaves.

[0044] Therefore, when 100 g of undried stems and leaves are added to extract solution A, the composition ratio of the extract solution is the same in both the Example and the Comparative Example. Specifically, the ratio of 5% acetic acid to ethanol is 5:95. In other words, the ratio of ethanol to acetic acid is 380:1. In the Example as well, the stems and leaves were immersed in extract solution A and stirred for 72 hours at room temperature in a dark environment.

[0045] For both the Example and the two Comparative Examples, 1000 μL of the extract was taken and the ethanol was evaporated using a centrifugal dryer. The precipitate was dissolved in 1000 μL of a methanol mobile phase mixture and passed through a 0.45 μm filter. 10 μL of the filtered solution was analyzed by HPLC (high performance liquid chromatography), and the α-tomatine content was determined from the peak. The HPLC measurement conditions are shown in Table 1. The amount of α-tomatine extracted from 100 g of tomato residue for the Example and the Comparative Examples, based on the HPLC measurements, is shown in Table 2.

[0046]

[0047]

[0048] Table 2 shows the amount of α-tomatine extracted from 100 g of moist tomato residue in both the Example and Comparative Example. The method of the present invention was able to extract an order of magnitude larger amount of tomatine than the Comparative Example.

[0049] Example 2: Repeated Extraction 200 g of tomato residue was prepared by combining tomato leaves and stems. An extract solution was prepared using a solvent of 3,800 mL of ethanol, 10 mL of water, and 10 mL of acetic acid. 200 g of tomato residue was added to this extract solution, and the mixture was stirred at room temperature in a dark environment for 72 hours. When 200 g of tomato residue had been added, the total volume of the extract solution was 4,000 mL. After stirring, the tomatine concentration in the extract solution was measured by HPLC.

[0050] The tomato residue was removed, and an amount of ethanol equal to the amount of liquid removed by the tomato residue was added to create a new extract. 200 g of tomato stem and leaf residue was added to the new extract, and the mixture was stirred at room temperature in a dark environment for 72 hours. From the second extraction, the water content in the extract increased. This procedure was repeated seven times. Including the first extraction, a total of eight extractions were performed.

[0051] The results are shown in Figure 2. Referring to Figure 2, the horizontal axis represents the number of times (times), and the vertical axis represents the tomatine content (mg) per 4 L of extract. Referring to Figure 2, the amount of tomatine increased at a nearly constant rate up to the fifth time. However, from the sixth time onwards, the amount of tomatine extracted decreased. During this extraction procedure, the volume of the extract was always maintained at 4 L, and the amount of tomatine increased with each operation, so it can be said that the tomatine in the tomatine-containing extract was concentrated.

[0052] Example 3: Dry Weight of Residue Five sets of 100 g of tomato residue were prepared, each having been produced for approximately the same amount of time, and an experiment similar to that in Example 2 was conducted. Similarly, five sets of 200 g of tomato residue were prepared, and a similar experiment was conducted using twice the amounts of water, acetic acid, and ethanol used in Example 2. After 72 hours of stirring, each sample (tomato residue) was separated from the extract, spread on absorbent paper, and left at room temperature for approximately 21 to 24 hours.

[0053] The tomato residues after standing were found to have almost no moisture remaining when inspected visually and to the touch. The weights (dry weights) of the tomato residues were measured. The results are shown in Tables 3 and 4.

[0054]

[0055]

[0056] Referring to Tables 3 and 4, the tomato residue had a weight of 8.78 g to 10.94 g per 100 g. In other words, the water content in the tomato residue varied from 91.22 g to 89.06 g. Therefore, when preparing the initial extract, the total water content may be 4% to 5%.

[0057] Example 4: Storage Characteristics The five-times-extracted example was subjected to storage tests at room temperature (23°C), low temperature (4°C), and freezing (-20°C). The tomatine concentration was measured after storage in solution for two months. The results are shown in Table 5.

[0058]

[0059] As shown in Table 5, the tomatine concentration decreased to about 72% at room temperature, but remained at 93.8% at low temperature and 89.5% when frozen, maintaining almost 90%. Therefore, it is recommended to store tomatine extract at or below low temperatures.

[0060] Example 5: Repellent Activity The repellent activity of the tomatine-containing extract obtained in this example against German cockroaches was confirmed as follows. An acrylic box measuring 30 cm x 30 cm x 30 cm was prepared as a test apparatus. The top of the test apparatus was opened for ventilation, and a nonwoven fabric was placed over it to prevent cockroaches from crawling out.

[0061] Two shelters, each made of plywood approximately 8 cm x 8 cm and a black translucent PVC board glued together with a 3 mm gap, were placed in the test apparatus. One shelter was used as the test area, and filter paper soaked in a tomatine extract and then air-dried was placed inside. The other shelter was used as the control area, and filter paper soaked in ethanol instead of the sample and then air-dried was placed inside.

[0062] Twenty adult German cockroaches were placed in the test device and allowed to stand overnight. The temperature was set at 25°C. The number of German cockroaches hiding in each shelter was then counted. This test was repeated three times. The results are shown in Table 6.

[0063]

[0064] Referring to Table 6, the "average" is the average of three tests for the test and control plots. This can be interpreted as the average number of invading individuals in the test plot and the average number of invading individuals in the control plot, respectively. In the test plot where filter paper impregnated with a tomatine-containing extract and then air-dried was laid out, not a single German cockroach invaded, resulting in a repellency rate of 100.0%, confirming sufficient repellency. The repellency rate was expressed as the ratio (percentage) of (average number of invading individuals in the control plot - average number of invading individuals in the test plot) to the average number of invading individuals in the control plot.

[0065] The method according to the present invention can be used to produce a tomatine-containing extract from tomato residue and can also be used effectively when industrially disposing of the tomato residue. This method kills two birds with one stone.

[0066] 1. Original tomato residue 2p: Extract (= precursor to initial extract) (with the water content of the original tomato residue subtracted) 2. Original extract (= initial extract) 2a: Original extract (from which tomato residue has been separated) 3. Original tomato residue (from which the water content has been replaced with ethanol) 4. (Dried) tomato residue 5. Ethanol (the same amount as the amount of liquid carried over by the separated tomato residue) 6. New extract 6a: Original extract (from which tomato residue has been separated from the new extract) 7. New tomato residue 8. Original tomato residue (separated from the original extract after the second time) 9. (Dried) tomato residue

Claims

1. A method for producing a tomatine-containing extract comprising the steps of: estimating the moisture content and dry weight of original tomato residue, which is the part of the tomato plant other than the fruit; and immersing the original tomato residue in an original extract having an ethanol to acetic acid ratio of 380:1, which is prepared so that the moisture content is 4% to 6% of the total.

2. A method for producing a tomatine-containing extract according to claim 1, comprising the steps of: withdrawing the original tomato residue from the original extract; estimating the amount of liquid that the withdrawn original tomato residue has carried from the original extract; adding ethanol to the original extract in an amount equal to the amount of liquid carried out to produce a new extract; and immersing undried tomato residue as a new tomato residue in the new extract.

3. A method for producing a tomatine-containing extract according to claim 2, comprising using the new extract as the original extract, using the new tomato residue as the original tomato residue, and repeating the steps of claim 2.

4. The method for producing a tomatine-containing extract according to claim 3, wherein each of the steps is repeated at least three times.

5. A method for treating tomato residue, comprising the steps of: estimating the moisture content and dry weight of original tomato residue, which refers to the parts of the tomato plant other than the fruit; immersing the original tomato residue in an original extract solution containing ethanol and acetic acid in a ratio of 380:1, which has been prepared so that the moisture content is 4% to 6% of the total; and removing the original tomato residue.

6. A method for treating tomato residue according to claim 5, comprising the steps of: withdrawing the original tomato residue from the original extract; estimating the amount of liquid that the withdrawn original tomato residue has carried from the original extract; adding ethanol to the original extract in an amount equal to the amount of liquid carried out to prepare a new extract; immersing the undried tomato residue in the new extract as a new tomato residue; and withdrawing the new tomato residue.

7. A method for treating tomato residue according to claim 6, wherein the new extract is used as the original extract, the new tomato residue is used as the original tomato residue, and the steps of claim 6 are repeated.

8. The method for treating tomato residues according to claim 7, wherein each of the steps is repeated at least three times.

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