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Physical, Chemical and Microbiological Characteristics of Tucuman Blueberry

Campero EV1, Gomez Marigliano AC2,3 and Barrionuevo MJ1*

1Department of Physics, Faculty of Exact Sciences and Technology, National University of Tucumán, Argentina

2Member of the scientific research career CONICET, Argentina

3Department of Physics, Faculty of Exact Sciences and Technology, National University of Tucuman, Independencia Avenue 1800, 4000, San Miguel de Tucuman, Argentina

*Corresponding Author:
Barrionuevo M Julia
Department of Physics, Faculty of Exact Sciences and Technology
National University of Tucumán, Argentina
Tel: 54-381 4364093
E-mail: [email protected]

Received date: 23/12/2016; Accepted date: 06/03/2017; Published date: 12/03/2017

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Blueberries are currently being studied because of the great interest generated by anthocyanins due to their potential health benefits due to their antioxidant activity for application in the food industry. In this work the physical and chemical properties of blueberries were studied. The results of antiradical activity were 23.23% in juice and 78.98% inshell; total polyphenol content was 279.17 ± 28 mg GAE/100 mL in juice and 447± 27 mg GAE/100 mL in shell. The values obtained in the microbiological analysis are similar to those obtained in other published works, so the technique allows us to predict the UFCs (Fungi) quite correctly. From the HPLC analysis, it is inferred that the juice of blueberries content the anthocyanins: cyanidin-3-galactoside, peonidin- 3-galactoside and malvinidine-3-glucoside. From the IR and Raman spectra it was found that the following polyphenols predominate: catechin, quercetin, kaempferol, galengina and morina.


Blueberries, Physical properties, Anthocyanin, Antioxidants


The "blueberry" is a berry almost spherical, its size can vary between 0.7 to 1.5 cm in diameter, light blue to dark in its interior containing up to 100 small seeds [1].

Since the 19th century the fruit of blueberries has been studied for its qualities for the prevention of urinary infections, water content, quinic acid, malic acid, citric acid, glucose and fructose [2]. In addition, the anti-inflammatory, anti-oxidant and antimicrobial properties due to their high content of polyphenols [3] adds an additional added value to the final product. Hence the importance of the conservation of the fruit that maintain its functional nutritional characteristics.

This fruit is in great demand in several regions of the world, particularly in the USA and Europe. It offers profits above those shed by traditional crops being more profitable than other traditional products (corn, sugar cane) [4].

The province of Tucumán is a major producer of blueberries for export. In the same the plantations extend in about 1,200 ha, which represents 46% of the total area of the country [5]. They lie on the Pedemonte, a transition strip between the western highlands and the depressed eastern lowlands.

Planted varieties are mainly O'Neal and Misty. The rest consists of an ensemble made up of other varieties such as Emerald, Jewel, Star, Bluecrisp. The production is made from September, concentrating the maximum in October and to a lesser extent in November.

This fruit has more than 80% water content and is a good source of fiber, vitamins and minerals; and various phytochemicals, mainly of phenolic nature, related to different parameters of organoleptic, nutritional and functional quality [6].

It is known that a diet rich in fruits and vegetables improves the general state of health and prevents cardiovascular diseases, neurodegenerative diseases, different types of cancer, etc. [7]. This protective effect seems to be associated to a great extent to the antioxidant capacity of different bioactive compounds, such as vitamin C and phenols, which are capable of preventing or slowing the oxidative processes involved in many pathologies [6].

For marketing CODEX-STAN 103-1981 requires that the product must be free of microorganisms and substances originating from microorganisms in amounts that may constitute a health risk. The Ministry of Agriculture, Livestock and Fisheries in its Quality Protocol establishes as Maturity Requirement differentiated maturity determined by content of sugars minimum 7° Brix, determined refractometrically.

It is used in juices, jellies, jams, drinks, etc. This generates a market with a growing demand because they are natural products with an industrial versatility that allows them to compete with other fruit juices [8]. In cranberry juices physical properties such as density, viscosity, refractive index, boiling rise and specific heat are affected by their solid content and temperature. Knowledge of physical properties is needed for the efficient design of food industry equipment, especially those requiring pumping of the product [9].

The viscosity of the blueberry juice is necessary to determine the heat transfer rates, the energy consumption with the increase of the concentration, and to control the temperature and the flows in order to ensure the continuous flow of the product [10].

The main objective of the work is to know the main components of the fruit, to evaluate the effectiveness of conservation methods and the possibility of extracting the feasible components to be used in derived industries, thus giving a greater added value to the local production of the fruit.


Anti-Radical Activity

Antiradical activity was determined in the shell and in the juice of cranberries preserved for 6 months at -18°C. For this purpose, the in vitro bioassay of the radical DPPH (1,1-diphenyl-2-picrylhydrazole) was used as reported in the literature [11], where radical capture capacity which have different extracts, by determining the degree of discoloration that they cause to an ethanolic solution of the radical.

A fresh ethanolic solution of the DPPH radical was prepared by weighing 2 mg of DPPH in a pre-weighed volumetric flask and dissolved in 100 mL of ethanol, reaching a final concentration of 20 mg/L covering the same from light. Ethanolic solutions of the compounds to be tested were prepared at concentrations of 100, 50 and 10 mg/L of both the shell and the juice. The DPPH solutions and ethanolic solutions of the samples and compounds tested were mixed, washed in a 30°C bath for 30 minutes in order to achieve uniform working temperatures after that time was read in a spectrophotometer at 517 nm.

The absorbance data for the extracts and / or compounds tested were replaced in the equation:

% Discoloration= (1 – Ac/ Ab)*100%

Where, Ac is the absorbance of the compound and Ab is the absorbance of the test blank (control).

From the percentage of discoloration the free radical scavenging capacity was determined. A value equal to 100 (one hundred) corresponds to the maximum free radical scavenging capacity, while a value close to 0 (zero) indicates a reduced or no capacity. The degree of discoloration indicates the efficiency of the substances extracted as radical scavengers in processes of oxidative stress. As a reference free radical scavenger, quercetin was used as a flavonoid with proven antioxidant activity.

Total Polyphenols

For the determination of total polyphenols, the modified micro-method of Folin-Ciocalteu (FC) [12] was used, which is based on the ability of the polyphenols to react with oxidizing agents. The Folin-Ciocalteu reagent contains molybdate and sodium tungstate, which react with any type of polyphenol, forming phosphomolybdic-phosphotungstic complexes. Electron transfer at basic pH reduces the phosphomonbital-phosphotungstic complexes in oxides, deep blue chromogens, tungsten (W8O23) and molybdenum (Mo8O23), this color being proportional to the number of hydroxyl groups in the molecule.

A calibration curve was constructed, using a standard solution of 500 mg/L (concentration) Gallic Acid. To 0.5 mL of a suitable dilution of each 0.3 mg/mL concentration sample, 0.5 mL of the Folin-Ciocalteau reagent was added and gently shaken, after two minutes 0.5 mL of 10% sodium carbonate solution and 3.5 mL of double distilled water. It was brought to the water bath for one hour at 30°C. After the time, measurements were made in the spectrum at 765 nm. The concentration of phenols in the samples was calculated on the basis of the calibration curve and expressed as mg equivalents of Gallic Acid / 100 g of sample.

Determination of the Composition of the Ntocianines of Bluberry Juice by HPLC

The composition of the anthocyanins from juice extracted from blueberry fruits was determined by waters brand HPLC with UV detector, octadecylsilane (C18) column of 250 mm in length and 4.6 mm in diameter, filled with particles of 5 micrometers in diameter, Using a mobile phase of methanol (60%) and water (40%).

Blueberryjuice Infrared and Raman Spectroscopy

Concentrations of 25, 50, 75 and 100% of the juice of whole fruits of blueberries in ethyl alcohol were used. The equipment used was FT IR Nicolet iS50 to determine the Infrared and Raman spectra ThermoScientific DXR Smart Raman.

Ultraviolet-Visible Spectrum

The UV-V spectrum was performed on a HITACHI U-1900 Spectrophotometer of the juices extracted from blueberry fruits, with dilutions in water at 100, 20 and 2% respectively.


Table 1 shows the physicochemical and microbiological determinations performed, the instruments and methods used, some of which have already been extensively explained in previous work group publications [13-15]. Others are briefly described below. UV spectroscopy, Raman and IR and HPLC chromatography assays were also performed to identify some major compounds and their composition.

Property Equipment Method
Density Digital Density Meter KEM DA-300 [16]  
Viscosity Viscosimeter ofOstwald [17]  
Refractive index Automatic Digital Refractometer Leica AR600 [17]  
° Brix Automatic Digital Refractometer Leica AR600  
Humidity Stove Method Oficial de AOAC 1995 990.20
Antiradical Activity Spectrophotometer HITACHI U-1900 "In vitro" bioassay of the radical DPPH (1,1-difenil-2-picrilhidracilo)
Total Polyphenols Spectrophotometer HITACHI U-1900 Modified Micromethod Folin -Ciocalteu
Total count of fungi   Surface sowing on potato dextrose agar, supplemented with chloramphenicol (merck) and incubating at 28°C for 5 days under aerobic conditions [18]
UV spectrum Spectrophotometer HITACHI U-1900  
Composition anthocyanins HPLC Waters, UV detector, Octadecylsilane (C18) column 250 mm long and 4.6 mm in diameter, filled with 5 micron diameter particles, using a mobile phase of methanol (60%) and water (40%).  
Raman Spectrum ThermoScientific DXR Smart Raman  
IR spectrum FT IR Nicolet iS50  

Table 1: Properties, methods and instruments used.

For the different trials, jewell variety blueberries were used from the Oran, Monteros, Tucumán “Tierra de Arándanos”Packing, preserved at -18°C for 180 days.

Results and Discussion

The effect of temperature on density at a constant concentration and the effect of concentration on density at specified temperatures have been studied by [8,9] for various fruit juices (Table 2).

T (ºC) d (kg/m3) η (Pa.s) I º Bx
15.00 1.0276 - - -
20.00 1.0264 - - -
22.00 - 0.99 - -
23.66 - - 1.35094 12
23.85 - - 1.35088 -
24.64 - - 1.35062 11.8
24.78 - - 1.35056 -
25.00 1.0249 0.989 - -
25.01 - - 1.35045 -
25.10 - - 1.35042 -
25.17 - - 1.35039 11.67
30.00 1.0233 0.945 - -
35.00 1.021 0.813 - -
40.00 1.0187 - - -

Table 2: Density values, viscosity, refractive index, Brix as a function of temperature.

In Figures 1-4 the variation of the property in function of temperature.


Figure 1: Variation of juice density in function of temperature. Line solid: linear equations, Dot: experimental values.


Figure 2: Variation of juice viscosity as a function of temperature. Line solid: linear equations, Dot: experimental values.


Figure 3: Variation of the refractive index of the juice as a function of temperature. Line solid: linear equations, Dot: experimental values.


Figure 4: Brix variation of juice as a function of temperature. Line solid: linear equations, Dot: experimental values.

In Table 3 we show the linear equations that were adjusted to represent the experimental data with R2.

Density d = -6.10-6 T2 - 10-5 T + 1.0292
Viscosity η = -0.0015 T2 + 0.0733 T + 0.1113
Refractive index n = -7.10-5.T2 + 0.003. T + 1.3187
Brix ºBx = -0.0273. T2 + 1.1138. T + 0.9215

Table 3: Equations of thermophysical properties as a function of temperature for the fruit juice of blueberries.

The studied variety presents total polyphenol contents similar to those found in the literature for cultivated blueberries of different origin. USDA Nutrient Database (2004) has published average values of 292.97 mg phenols / 100 g fresh blueberry for the most consumed varieties in the USA. In a paper Ochmian et al. [19] concludes that the phenolic content at best is 231.03 mg total phenols/100 g of fresh blueberry harvested in Poland. Giovanelli et al. [20] has found that the phenolic content values for blueberries harvested in Italy range from 250 to 310 mg phenols/100 g fresh blueberry (Tables 4-6).

Ethanol Extracts   Concentration of Extracts Total Phenols (mEAG / 100 g sample)
(100 µg/ml) (50 µg/ml) (10 µg/ml)
Juice 23.23% 11.98% 3% 279.17 ± 28
Shell 78.98% 50.71% 8.33% 447 ± 27

Table 4: Values of antiradical activity at different concentrations and total content of phenols in the juice and in the husk of blueberries.

  Insoluble dietaryfiber (FDS) Dietary fiber soluble (FDS) Dietary fiber Total (FDT) % Humidity at 55 °C % Ash UFC /g of fruit
Complete Fruit 1.54 ± 0.05 0.53 ± 0.02 2.07 ± 0.07 85.59 1.78 6200

Table 5: Values of dietary fiber, moisture, ash and CFU of blueberry fruits.

Name of anthocyanin % anthocyanins
Delfinidine 3-galactoside 2.9
Delfinidine 3-glucoside 1.5
Cyanidin 3-galactoside 68.3
Cyanidin 3-glucoside 2.8
Cyanidin 3-arabinoside 3.4
Peonidine 3-galactoside 12.2
Malvinidine 3-glucoside 3.9

Table 6: Total anthocyanin content of blueberry fruits obtained by HPLC.

Anthocyanins are characterized by an electron deficiency due to their particular chemical structure. which makes them very reactive to the free radicals present in the body. Consequently they may be potent natural antioxidants [21]. The properties attributed to anthocyanins to improve health are associated with this ability to act as antioxidants and sequester free radicals in biological systems. They can donate hydrogens or electrons to free radicals or trap and displace them in their aromatic structure [22].

The content of anthocyanins obtained with HPLC differ with respect to the data observed in literature [22]. Although maintaining the proportion in which they are found. The predominant anthocyanins being cyanidin-3-galactoside. peonidin-3-galactoside and malvinidine-3-glucoside (Figure 5).


Figure 5: Raman spectra of solutions to different % v/v the juice of blueberries in ethanol. 100% continuous line; ... 25%; ---- 50%; and -.-.-. 75%.

It is observed in Table 7 in accordance with Figure 5 that infrared spectroscopy shows signals that characterize the presence of aliphatic chain. as well as the presence of carbonyl compounds such as aldehydes, ketones or carboxylic acids in addition to the signal characteristic of the benzene derivatives [23].

Bond Compound Type Frequency Range (cm-1) Intensity
C-H Alkane 2800-3000 Low
C=O Aldehyde ketone-acid-carboxylic acid 1500-1600 Low
C-O Substitutions in aromatic ring 850-1100 Strong

Table 7: Frequencies of functional groups in the infrared spectrum/raman of the fruits of blueberries.

According to the authors Domingo et al. [24] we observe that the Raman spectrum (Figure 5) would indicate that the peaks 200, 400, 800, 1000, 1100, 1200, 1300 identifies catechin; 800, 900, 1100, 1500 quercetin; 400, 800, 1500, 1600 kaempferol; 500, 600 galengina and 400,1500 morina in the blueberry juice.

UV-Visible Spectra

The polyphenols absorb in the ultraviolet (UV) region. In the case of flavonoid-type phenols there are 2 characteristic absorption bands [25] the band of the aromatic ring A with a maximum absorption in the range 240-285 nm (benzoyl band) and another band of ring B with maximum of Absorption in the range 300-550 nm (Cinamoil band) (Figure 6).


Figure 6: Ultraviolet-visible spectrum of solutions to different % v/v the juice of blueberries in waterl. 100% continuous line;- - - - 20%; and --- 2%.

Visible ultraviolet analysis of blueberry juice at 100%, 20%, 2% dilutions in water showed a maximum absorption band (composite structure) between 200 and 300 nm which corresponds to the benzoyl or band II band characteristic. The aromatic ring A of flavonoids with peaks at 384, 366 and 322 nm and another very wide band at 520 nm which is the I band or cinamoil band and corresponds to the aromatic ring B of the flavonoid structure. The displacement of this last band at wavelength greater than 500 nm is characteristic for flavonoids of the anthocyanin group [26-29] thus verifying the presence of anthocyanins in addition to other phenolic substances as shown in Figure 6. The absorption band at 515 nm is characteristic for anthocyanins.

In the 0.2% spectrum a peak is clearly seen at 338 nm. In the 2% the band can be clearly seen at 538nm and 475 nm (less intense). At lower dilutions the band of 384 becomes very evident.

Figure 7 shows a band fitting that allows to conclude what type of bands are present and how they change with the dilution process.


Figure 7: Curve fitting of the Ultraviolet-Visible Spectrum of thejuice of blueberries.■. experimental band; continuous lines. Cumulative Fit Peak; ___ - - -. ...... -.-.-.. ----- and - . - -. bands obtained in the fitting process: 304. 333. 345. 460 and 538 nm.


The shell has a greater amount of polyphenols as well as a higher capturing capacity of the DPPH radical compared to the juice. The content of total polyphenols shows values similar to those published in the literature. The values obtained in the microbiological analysis are similar to those obtained in other published works. so the technique allows us to predict the UFCs (Fungi) quite correctly. From the HPLC analysis it is inferred that the juice of blueberries possesses the anthocyanins: cyanidin-3- galactoside. peonidin-3-galactoside and malvinidine-3-glucoside. From the IR and Raman spectra it was found that the following polyphenols predominate: catechin, quercetin.


The authors are grateful for the support provided by Consejo de Investigaciones de la Universidad Nacional de Tucumán of Argentina (Project nº 26/E516; 26/E505).