*2.4. Preserving Juice*

The juice obtained in the first harvest term (at seven days after plant emergence) was additionally subjected to pasteurisation for 10 min at 75 ◦C and freezing. Immediately after the thermal treatment, the product was cooled (in a blast chiller–freezer) to 3 ◦C. The other batch of juice was blast-frozen to a temperature of −18 ◦C. The material was stored under appropriate conditions, i.e., 4 ◦C in the case of the unprocessed (UJ) and pasteurised (PJ) juice and −18 ◦C in the case of the frozen juice (FJ). The study material was analysed after three (UJ and PJ) and seven (UJ, PJ, and FJ) storage days.

#### *2.5. Determination of pH*

Juice samples were placed in 50 mL beakers and the pH was recorded with a pH meter (model 780, Metrohm AG, Herisau Switzerland).

#### *2.6. Measurement of Total Protein Content*

The determinations were carried out using the Kjeldahl method and a Foss Kjeltec 8400 automatic distiller (Foss Anatytical AB, Höganа¨s, Sweden). The total protein content was calculated using a 6.25 conversion factor.

#### *2.7. Determination of the Chloride Content*

The chloride content was determined with the Mohr method using a TitraLab AT1000 Series automatic titrator (HACH Company, Willst¨аtterstraße, Germany). The solution was titrated with a 0.1 N silver nitrate solution. The chloride content was given as g in 100 gf.j. (of fresh juice).

#### *2.8. Determination of the Content of Reducing Sugars*

The Lane–Eynon method was used to determine the content of reducing sugars. The material was extracted and deproteinised. The content of reducing sugars was determined in the obtained liquid by direct hot titration of a specific copper salt with the analysed sugar solution (against methylene blue as an indicator of the end of the reaction) [15,16].

#### *2.9. Determination of Dry Matter Content*

The moisture content of the research material was measured by drying 3 g of juice at 105 ◦C for 3 h. The measurements were carried out in triplicate.

#### *2.10. Measurement of Chlorophyll Content*

The juice was analysed for the content of chlorophylls A and B. The pigments were extracted with methyl alcohol. The chlorophyll content was measured using a UV–vis Helios Omega 3 spectrophotometer (Thermo Scientific, England). The measurement consisted of the determination of the absorbance (A) at different wavelengths (λ): 650 and 665 nm [17]. Next, the content of chlorophylls A and B and the total chlorophyll content were calculated with Equations (2)–(4):

Chlorophyll A content (Cchl(a)):

$$\mathbf{C\_{chl(a)}} = 16.5 \cdot \mathbf{A\_{(665)}} - 8.3 \cdot \mathbf{A\_{(650)}} \tag{2}$$

Chlorophyll B content (Cchl(b)):

$$\mathbf{C\_{chl(b)}} = \mathbf{33.8} \cdot \mathbf{A\_{(650)}} - \mathbf{12.5} \cdot \mathbf{A\_{(665)}} \tag{3}$$

Total chlorophyll content (C):

$$\mathbf{C} = \mathbf{4.0} \cdot \mathbf{A}\_{\text{(665)}} + \mathbf{25.5} \cdot \mathbf{A}\_{\text{(650)}} \tag{4}$$

where:

*A(650)* = absorbance at a 650 nm wavelength;

*A(665)* = absorbance at a 665 nm wavelength.

The chlorophyll content was calculated in mg·g<sup>−</sup>1, taking into account the sample weight.

#### *2.11. Statistical Analysis*

The data were analysed statistically. A significance level of α = 0.05 was assumed for inference. The analysis was carried out using ANOVA (StatSoft Polska, Poland) with post hoc tests for homogeneous groups based on Tukey's test. These groups comprised means between which no statistically significant difference was found at the assumed significance level, α.

The determinations were carried out in triplicate, except for leaf length and strength tests, which were repeated five times.

#### **3. Results and Discussion**

#### *3.1. Characterisation of the Physical Traits of the Raw Material*

Changes in the length of the barley shoots relative to the harvest date are shown in Table 1.

**Table 1.** Properties of the raw material relative to the harvest time of barley leaf harvesting.


a,b,c,d Means in the same line denoted by different letters were significantly different. The results are expressed as mean ± SD (*n* = 5).

The largest gain in the length of barley shoots was noted within seven days after emergence. In the following days, the rate of shoot growth declined. There were no statistically significant differences in the length of shoots between the material harvested at day 21 and day 28 after emergence. The length of shoots collected on day 21 and day 28 was 17.58 and 19.53 cm, respectively. The height of the plants was characteristic of unfertilised crops [18–20].

The strength of the barley shoots decreased over time. However, there were only significant changes in the tensile strength of the shoots in the material collected on day 28 of growth (in comparison to the material collected on day 7). Changes in the strength of cereal shoots are associated with the chemical composition, which is modified during plant growth [18].

#### *3.2. The Pressing E*ffi*ciency*

The effect of the harvest date on the pressing yield is shown in Figure 2.

**Figure 2.** Pressing yield of the barley shoot and leaf juice relative to the harvest date.

The pressing yield ranged from 69.04% to 73.26%. It was obtained from 137 to 146 mL of juice (depending on the time of harvest). The highest value was noted during the processing of material harvested on cultivation day 21. The longer period of cultivation was associated with a statistically significant drop in the pressing yield. The pressing yield of barley leaves collected on day 28 was estimated at 69.4%. These results are consistent with data obtained by other authors. Paulíckov ˇ á et al. [2] reported a pressing yield of 68% in a study that involved the extraction of juice from barley shoots. The decline in the pressing yield accompanying the longer harvesting period is probably caused by changes in the chemical composition, which lead to an increase in the fibre content.

#### *3.3. Juice Acidity*

Irrespective of the harvest date, the juice from the leaves and shoots of young barley had an acidic reaction (changes in pH are shown in Figure 3), shown by the significant decrease in pH values over time. The pH of the products pressed from leaves and shoots collected on day 7–28 ranged from 5.71 to 5.95. These values are similar to the pH of vegetable juices such as carrot or beetroot juice [21–23].

**Figure 3.** Impact of barley shoot harvesting time on juice pH.

The pH values of the juice from young barley leaves did not change significantly during storage (Figure 4). The statistical analysis only revealed significant differences in the pH value in the case of juice refrigerated for seven days. The pasteurisation and freezing processes did not change this parameter significantly. Juice acidity has an important effect on pigments and other ingredients (e.g., chlorophyll, carotenoids, anthocyanins, myoglobin, etc.) responsible for the colour of fruits, vegetables, and meat [21,24–26].

**Figure 4.** Impact of the conditions and length of storage on barley juice pH. (CP—control probe for fresh juice, UJ—unprocessed juice, PJ—pasteurised juice, FJ—frozen juice; 3rd day—after three storage days, 7th day—after seven storage days).

#### *3.4. Total Protein Content*

Protein content was expressed as a percentage of dry matter, which was 4.71% on average. Changes in the protein content of the analysed juices are shown in Figure 5. The total protein content in the juice increased along with the barley harvest date. The differences in the protein content between the harvest dates were statistically significant. The highest protein content, i.e., 51%, was determined in samples collected on day 21. The statistical analysis revealed that the content of this component in the juice produced after the next harvest (day 28) was significantly lower (42.68%). As demonstrated by Paulíckov ˇ á et al. [2], the total amino acid content in juice from barley leaves and shoots decreased over time. The highest protein content recorded for the Malz cultivar (collected in phase I—DC 29) was 30.44%d.m. However, it should be noted that the authors collected the raw material at a later stage of barley growth. Therefore, these results may explain the lower protein content in the juice from barley leaves and shoots harvested on day 28 of growth (in comparison with the material obtained on day 21).

**Figure 5.** Changes in the protein content in barley shoot juice relative to the harvest date.

### *3.5. Chloride Content*

The changes in chloride content in the analysed juices are shown in Figure 6. The chloride content in the juices was positively correlated with the length of barley growth. The statistical analysis demonstrated statistically significant differences in this parameter between the harvest dates. The chloride content ranged from 0.021 to 0.117 g·100 g f.j. <sup>−</sup><sup>1</sup> for juice pressed from barley leaves and shoots collected on days 7–28. The increase in the chloride content was clearly correlated with a decrease in the pH of the juice. Park et al. [27] also showed that the amount of chlorides in the aboveground parts of plants may depend on the type of fertilization used.

**Figure 6.** Changes in the chloride content in barley shoot juice relative to the harvest date.

#### *3.6. Content of Reducing Sugars*

Changes in the content of reducing sugars are shown in Figure 7. Their highest content was determined in the juice from barley leaves and shoots collected on day 21 of growth (8.20 g·100 gd.m. <sup>−</sup>1). The differences in the value of this parameter between products obtained from shoots collected on days 14 and 28 were not statistically significant. The statistical analysis confirmed the lower content of reducing sugars only for juice made from raw material collected on day 7. The study conducted by Paulíckov ˇ á et al. [2] showed that the content of simple sugars varied, depending on the plant growth phase. Other factors that significantly determined the changes in the analysed parameter include the conditions of the soil and the variety of barley. Paulíckov ˇ á et al. [2] demonstrated that the sugar content in most barley varieties steadily decreased throughout the growing season.

**Figure 7.** Changes in the content of reducing sugars in barley shoot juice, depending on the harvest date.

#### *3.7. Chlorophyll Content*

The chlorophyll content in the barley juice and the impact of the thermal treatment methods on changes in this parameter are shown in Table 2. The content of chlorophyll A and B and the total chlorophyll content changed significantly during storage. The highest determined content of total chlorophylls, i.e., 6.62 mg/g, was found in fresh juice. The chlorophyll contents in fresh raw material obtained by other authors ranged from 10.15 to 19.62 mg/g [28,29]. After seven days of storage, the total chlorophyll content was reduced by 37.9% (UJ), 42.12% (PJ), and 2.43% (FJ), in comparison with the untreated juice. It can thus be concluded from the present investigations that chlorophyll A is more sensitive to heat than chlorophyll B. Similar findings were reported by Weemaes et al. [30], who analysed the kinetics of chlorophyll degradation in thermally treated broccoli juice.

**Table 2.** Changes in the chlorophyll content in juice from young barley leaves during storage in various conditions (*p* < 0.0001).


a,b,c,d,e,f Means in the same line denoted by different letters were significantly different. The results are expressed as mean ± SD (*n* = 3).

The process of freezing had the lowest effect on changes in the content of chlorophyll A and total chlorophylls. By contrast, in the case of chlorophyll B, the least significant changes were noted for the unpasteurised product refrigerated for one day and in the case of the frozen juice stored for seven days. Paulíckov ˇ á et al. [2] also confirmed the significant effect of thermal treatment of barley leaf juice (freezing, drying, and freeze-drying) on changes in the nutrient content. The analysis of their results also allows for the conclusion that freezing exerts the weakest effect on the quality of the product. Koca et al. [25] demonstrated a faster rate of degradation of chlorophylls at a lower pH value (from 5.5 to 7.5). Hence, the significantly lower chlorophyll content of juice stored for seven days may be caused by, e.g., changes in the pH of the product.

### **4. Conclusions**

The present investigations confirm the potential benefits of consuming green juice from young barley shoots and leaves as part of a daily diet. The product obtained from the material harvested after 7, 14, 21, and 28 days of growth contains many valuable nutrients, e.g., a high level of total protein (with CS = 41.44 Meth). Additionally, high pressing yields of approximately 70% can be achieved.

The study has demonstrated that barley leaves and shoots harvested on day 21 of plant growth are the best raw material for the production of juice. The process of pressing material collected at this time exhibits the highest efficiency, and the juice contains the highest levels of protein and reducing sugars, as well as a high chloride content.

The most effective way to preserve the juice from young barley leaves is freezing. This process does not induce changes in juice acidity and only slightly reduces the content of chlorophylls A and B during storage of the product. Pasteurisation of the juice significantly reduces the chlorophyll content, but does not induce statistically significant changes in the pH of the juice.

The reported results are a preliminary study on the topic. However, it is necessary to continue research to determine the impact of other factors (e.g., barley varieties, growing conditions) on the quality of juices from young barley shoots and leaves. It is also possible scale-up the experiment using a higher number of samples, filed conditions, etc.

In addition, this way of using shoots and leaves of young barley will have economic importance. The costs of obtaining raw materials for juice production will be reduced. The use of shoots and leaves of young barley, which is grown as a forecrop, for the production of green juices, may have a beneficial effect on the development of sustainable crop production. This will allow more efficient use of the plants.

**Author Contributions:** Conceptualization, A.B.-K., D.A., F.K. and L.R.; methodology, A.B.-K. and D.A.; formal analysis, A.B.-K. and F.K.; investigation, A.B.-K. and L.R.; data curation, A.B.-K. and L.R.; writing—original draft preparation, A.B.-K. and Z.K.; writing—review and editing, D.A.; visualization, A.B.-K.; supervision, D.A.

**Funding:** This research received no external funding.

**Conflicts of Interest:** The authors declare no conflicts of interest.

#### **References**


© 2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

*Article*
