Materials and Chemicals 1. Material: Petit vert

Petit vert was supplied in frozen form from Kagome Co., Ltd., Japan. The sample was stored under frozen conditions. GPC-grade solvent, tetrahydrofuran (THF) was purchased from Wako Chemicals (Japan).

Petit vert is a new vegetable that was born in Shizuoka, 1990. In French, the meaning of Petit vert (プチヴェール) is “small green”. What a lovely name! In late years although it grew to come out at limited store, that "small green" isn't familiar vegetable like a lettuce and a cabbage. Petit vert is a new vegetable which is developed by crossing kale with cabbage when continuously cultured in farm field with poor drainage (Fig. 4.1). It is world's first non-head nature Brussels sprouts and it inherits a good point of parents, and it has nutritive value more than kale, and to include taste of Brussels sprouts.

As a result of the research and development that demanded high functional vegetables of nutritive value from the first, Petit vert was invented such a new vegetable now. By now, because it has high quality nutritional ingredients, antioxidant functions Petit vert has a great attention.

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Figure 4.1. Petit vert is a hybrid vegetable.

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Basic physical structure of petit vert is explained in Fig. 4.2 below.

Figure 4.2. Structure of Petit vert.

Outside leaf

This part has much more nutritive values than that of bud of kales. It is abundant in calcium and particularly in carotene. It does not appear in the market as a green leaf, it is actively found as juice or a supplement.

Main body

Although the total image gives a feeling like a bud, it is slightly strange, has a little bit more strength.

The height of the tree changes between 50-60 cm. cultivation is done when the length of 80 cm is reached.

It seems to be little low when the season is in dry trend.

Stem

Side bud grew thickly is to the stem.

One bud sticks to one piece of leaf, and about 30-50 PetitVert sticks to one bowl (by cultivation in a field, about 50-80).

Bud

It does not have a head like cabbage in form as rose. It is ready to be eaten if it becomes 3-5 cm in diameter.

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Most of high quality nutrients were inherited to Petit vert from kale and Brussels sprouts. The nutritive value exceeds kale, and its properties are much more similar with Brussels sprouts. Besides, iron which shows deficiency in women is mostly found more than cabbage in Petit vert, so that it took the name as top-class among vegetables. It has a quantity of more than 2 times of parents’ in iron content. Vitamin C exists to make iron absorption, and it is an all-around player in the body.

Figure 4.3. Comparison of nutritive values of various foods with Petit vert.

Because high content of vitamin C, carotene, iron and calcium content has been particularly superior in balance of nutritive value, lack of vegetables can be easily supplied by Petit vert and the most important is, it has valuable components that are crucial for height and balance assets. In particular, its chemical composition was

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comprised with other vegetables in Figs. 4.3 and 4.4. As a result of nutritional analysis, the contents were explained for Petit vert on 100 grams basis as illustrated in Table 4.1 below.

Figure 4.4. Chemical composition similarity of Petit vert with other vegetables.

Table 4.1. Basic chemical components of Petit vert.

Dietary fiber (g)

Calcium (mg)

Iron (mg) Caroten (µg)

Vitamin C (mg)

Petit vert 6.7 457 2.2 4,320 140

Brussels sprouts

1.4 35 1 400 150

Kale 3.7 220 0.8 2,900 81

Cellulosic fiber

Vitamin C Calcium

Caroten Iron

Petit vert

kale

Brussels sprouts

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As for the experimental studies, mainly hydrothermal treatment was chosen as general method. Because of nourishing properties of Ca²⁺ ion, a method for the extraction was tried to be optimized. On the other hand, K⁺ ion was taken into consider as an indicator for pectin recovery.

The quantity and the quality of pectin are varied considerable depending on the kind and the part of plants, though the pectin exists universally in the cell wall and the middle lamella structure of all higher plants [8]. It is well known that jam and jelly are manufactured by using the gel formation property of the pectin. The pectin can be extracted and separated by using mild acid in industry. Most of these pectin products are used for the production of jelly, jam, marmalade, and confectionery. Pectin is composed of the acidic polysaccharide and several kinds of neutral sugars [7, 9]. Main chain consists of α-1, 4-linked D-galacturonic acid, which is partly methyl esterified  as shown in Fig. 4.5.

Figure 4.5. Main chain of pectic substance.

The non-esterified galacturonic acid units can be either free acids (carboxyl groups) or salts with sodium, potassium or calcium. The salts of partially esterified pectins are called pectinates, if the degree of esterification is below 5% the salts are called pectates, the insoluble acid form, pectic acid [8].

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Figure 4.6. Assumption structure of protopectin in plant.

It is thought that it is impossible to take out the pectin in the cell tissue as it is.

Because Protopectin units form an insoluble macromolecule with cellulose or form an insoluble salt called pectinate with Ca and Mg. Henglein [6] advocates the hypothesis like Fig. 4.6 about the structure of protopectin.

The method of excluding the cation that constructs the pectic acid mutually by using chelator and cutting the hydrogen bond with the cellulose by using the acid has been used for the extraction of protopectin so far as understood from Fig. 4.6.

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Pullulan standard kits were acquired from Shodex Company (Shodex STANDARD P Series- P-82, Shodex Ltd., Tokyo- Japan). GFC solvent, pure water was homogenized using ultrasonic cleaner for 15 minutes and centrifuged for 30 minutes.

4. 2. 2. Experimental Procedure

Subcritical water extraction, that is, extraction using hot water under pressure sufficient to maintain water in the liquid state, has demonstrated its ability to selectively extract different classes of compounds depending on the temperature used. The selectivity of subcritical water extraction allows for manipulation of the composition of the extracts by changing the operating parameters [5, 9, and 10].

Figure 4.7. Subcritical water extraction flow diagram.

T2

Water

Pump

Heater

Heat Exchanger

Filter BPR

Sampling T1

T3 P

Extractor

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Extraction mechanism which is named as semi-continuous type subcritical water extraction was used. This kind of water treatment was done in order to emphasize the effect of water mobility in the subcritical extraction process. For semi continuous scale subcritical water extraction, a schematic diagram of a laboratory built subcritical water extraction system whose operating flow diagram is presented in Fig. 4.7. The system consists of two HPLC pumps (PU-2080-100 MPa, Jasco Co., Japan), an oven, a stainless steel extraction cell (10 ml Vessel, Thar Designs, Inc., USA), and a collecting flask.

Water was de-oxygenated for 30 min using ultrasonic cleaner (Honda, W-211) prior to the extraction. With the HPLC pump, the water was then delivered at a constant flow rate to the extractor and the extraction cell was completely filled with plant material (3 g of Petit vert) and mounted vertically in the oven with water flowing from top to bottom.

The water was brought to a set temperature by means of the preheating coil inside the oven before entering the extractor. The extraction pressure was controlled by adjusting the back-pressure regulator (AKICO) connected to the outlet coil.

A chiller (Shibata Co., Japan) was used to cool the extract from the oven to a constant temperature close to 25°C, thus avoiding losses of the products caused by the hot water. Different extraction procedures have been used depending on whether individual extractions (at a chosen temperature). The extraction experiments were carried out mainly to determine the effect of temperature holding the pressure and water flow rate constant. The effect of temperature in the extraction process was studied. For this purpose, different assays were carried out. Among them, samples were extracted once with water at constant temperature being 100, 150, 175 and 200°C respectively working at pressure around 10 MPa to keep the water in the liquid state. The flow rate used was 1 ml/min for maximum 310 minutes of experimental time. After that, in order to observe the effect of gradually increasing temperature on extraction mechanism, the experimental temperature conditions were changed to around 25°C up to 200°C increasing gradually, keeping all remaining parameters constant. The temperature diagrams for the experiments were given in Figs. 4.8 and 4.9 in which temperature was constant and was increased gradually. All runs were performed at least in duplicate. Any doubtful results were checked and the experiments were repeated up to five times.

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Figure 4.8. Temperature profile for experiments at constant temperatures.

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Figure 4.9. Temperature profile for experiments at gradually increased temperatures.