Preparation and chemical characterization of banana / orange composite wine

Banana/orange composite wine was brewed in the lab by liquid fermentation using angel yeast and lactic acid bacteria as the fermentation strains, and characterized by chemical analyses. It was found that the best ratio of banana juice to navel orange juice was 1:2, and the optimum alcohol fermentation parameters were as follows: 28~30°C, 22% initial sugar content, 6% yeast, and 6 days of fermentation to reach an alcohol concentration of 11.63% v/v. The free amino acids in the composite wines were tested by automatic amino acid analyzer, and the flavor components of the composite wine were determined and analyzed by gas chromatography-mass spectrometry (GC-MS). Seven types of trace elements in the composite wine were measured by atomic absorption spectrometry. Results showed that there were 17 free amino acids, and their total concentration was up to 897.6 mg/L. A total of 16 key compounds were identified in the composite wine, 11 of which were ester, 4 of which were alcohols and 1 of which was acid. Magnesium, iron, copper and manganese elements were relatively rich in the wine, while lead was extremely low.


INTRODUCTION
2][3][4] Apple, kiwifruit, jujube, peach and lychee wine have been developed in China in recent years.With the improvement of living standards, people have put more attention to the health care function of fruit wine.For two or more composite fruit wine brewing, it probably keeps a variety of fruit flavor, making wine more nutritional value and palatability, and own good healthcare function.Meanwhile, the development of composite fruit wine conforms to the trend of wine because of the abundant fruit resources in China. 5,6However, composite fruit wines are mainly researched in the stage of laboratory.
The co-culture of lactic acid bacteria and yeast stable is widespread in food processing, the acidic environment created by lactic acid bacteria has a positive effect on the proliferation of yeast.Meanwhile, many growth factors in yeast, such as vitamin, soluble nitrogen compounds, also promote the growth of lactic acid bacteria.Adding lactic acid bacteria during alcoholic fermentation of fruit wine can trigger the malic-lactic acid fermentation.As a result, the malic acid changes into lactic acid after decarboxylation reaction, which could make wine smooth and taste flavor and fragrance. 7][10][11][12][13][14] However, banana and navel orange composite wine has not been reported by now.In this paper, brewing technology of banana and navel orange composite wine was studied.The yeast and lactic acid bacteria were used as mixed ones for fermentation.GC/MS, high performance liquid chromatography (HPLC) and atomic absorption spectra were used to investigate the content of flavor compounds, amino acids and trace elements to determine the optimal parameters.

Materials
Banana and navel orange was purchased at Maoming fruit market without plant diseases.Angel yeast was purchased from Angel Yeast Co., Ltd.Lactic acid bacteria was purchased from Beijing Chuanxiu Technology Ltd. 17 types of amino acid standard solution were purchased from the Waters Company.The standard solution of calcium, iron, magnesium, manganese, copper, lead and cadmium were provided by the Analysis and Test Center of Guangdong University in Petrochemical Technology of Guangdong University.

Banana and navel orange composite wines brewing process
Scheme 1 presents the brewing process of banana and navel orange composite wines briefly.Fresh bananas were peeled, cutting into 2 cm thick slices, and heated in the hot water at 90 °C for 3min.After cooling and filtering water, bananas were broken with water by the mass ratio of 1:1.5, then added with 0.05% pectinase (Wt ratio, based on banana).The enzymatic hydrolysis was processed at 50 °C for 2.5 h.Finally, the solution was filtered to obtain banana juice.
Fresh navel oranges were sorted, cleaned, peeled and pressed.Then enzyme was inactivated in water bath at 85 °C for 15 min to obtain navel orange juice.Banana juice and navel orange juice were mixed with 1:1 weight ratio.Sucrose was used to adjust the mixed juice sweetness.After ORIGINAL PAPER activated, yeast and lactic acid bacteria (2:1 ratio) were added into the mixed juice, and then fermented at 25~35 °C .
Fermentation conditions are as follow: temperature was 26, 28, 30, or 32 °C ; sugar percentage was 16, 18, 20, or 22%; fermentation time was 3, 4, 5, or 6 d; inoculation amount of yeast and lactic acid bacteria (2: 1 ratio) was 5%, 6%, 7% or 8%.These factors were assumed the impact factors to determine the best conditions of alcohol fermentation with orthogonal test.High performance liquid chromatography (HPLC) was used to determine the content of amino acid in the composite fruit wines.The sample was derived with phenyl isothiocyanate and dried in vacuum, then was dissolved with phosphoric acid acetonitrile solution (pH 7.4).The supernatant was tested in HPLC after centrifugation.The column temperature was 38 °C .Absorbance was monitored at 254 nm at a flow rate of 1 mL/min.(Amino acid analysis column, PICO.TAG, 3.9 mm × 150 mm) Trace elements in the composite fruit wines were tested by atomic absorption spectrometry.The tested sample was obtained as fallow.The 5 mL wines sample was removed alcohol on heating apparatus at 150 °C , and 10 mL concentrated nitric acid was added at the same temperature.Then, the sample was put into the microwave digestion system for digestion until 1~2 mL sample solution remained with adding 2 mL concentrated nitric acid and 1 mL hydrogen peroxide, then diluted to 50 mL.The working conditions of this instrument are showed as Table 1.

Optimization of alcoholic fermentation conditions
As shown in Table 2, the optimal technological conditions for fermentation temperature was 28 ~30 °C .Yeast and lactic acid bacteria inoculation amount were about 6%.The sugar concentration reached 22% and the fermentation time was 6 d.The impact sort of various factors on ethanol fermentation was: sugar concentration > fermentation time > fermentation temperature > inoculation amount.Table 3 showed the P value of initial sugar concentration was less than 0.01, which indicated that the initial sugar concentration played a very important role in alcoholic fermentation while inoculation and fermentation temperature showed no significant effect.
Because the optimal combination conditions did not emerge in the orthogonal test, verification test was processed with the 28 °C of fermentation temperature, 6% of inoculation amount, 22% of sugar and 6 d of fermentation time.As a result, alcohol concentration of composite fruit wine was 11.63% (v/v).This condition could be the optimal one.Fig. 1 showed GC-MS total ion current chromatogram of banana and navel orange wine component.By contrasting similarity (SI) of NIST mass spectrometry database, the flavor components of banana and navel orange composite wines were identified.As is table 4 shown, the relative content of each component was calculated by peak area normalization method.The retention time was below 24 min because longer time led to higher temperature which would give rise to lower similarity.As a result, 16 compounds which similarity (SI) was more than 80 were identified according to MS NIST database in banana and navel orange composite wine, and these 16 compounds possessed 96.15% of the total peak area, including 4 types of alcohols accounted for 79.43%, 11 types of ester for 15.14%, 1 type of acid for 1.58%.Alcohols and esters were the main flavor components of the composite wine.Wine flavor is divided into three categories: the varieties of fruit, fermentation, and ageing.In general, the main flavor components in fruit wine produce in fermentation 11 .Alcohols are the most important substances in the composite fruit wine, which was derived mainly from the wine fermentation process.Wine containing adequate higher alcohols possess special flavor, such as benzene ethanol (0.266%) with sweet rose, violet, and jasmine fragrance. 12Esters are main flavor components in banana and navel orange composite fruit wine, such as ethyl caprylate (6.521%) with rose and orange fragrance; ethyl acetate (3.221%) and ethyl benzoate (0.404%) with fruit fragrance; isoamylacetate (2.242%) with banana and apple fragrance; hexyl acetate (0.39%) with pear and apple fragrance; benzene ethyl acetate (0.372%) with rose and apple fragrance.Consequently, these alcohols and esters prompt the composite fruit wine possessing a special, pleasant aroma.

Determination of the content of amino acid in banana and novel orange composite wine
The content and composition of amino acid is one of the important indicators on the quality of the composite fruit wine.Amino acids derive from protein enzymolysis in fruit juice, metabolites of yeast fermentation and after fermentation of yeast cells.The presence of amino acids has an important effect on the taste of wine. 13Table 5 showed that 17 types of amino acids were investigated in banana and novel orange composite fruit wine, which included 7 types of essential amino acids (except tryptophan) accounted for 21.51% of total amino acids.The content of proline was the highest with 32.29% among them.Amino acids play an important role in wine aroma.Sweet taste of fruit wine was contributed to proline, serine, glycine, alanine, histidine and leucine.Especially, proline presences both sweet and bitter, which are responsible for the complexity of wine taste and flavor. 13

Determination of trace elements in banana and navel orange composite wine
Trace elements participate in the formation of a number of enzyme, coenzyme, vitamins, hormones and nucleic acid with a specific physiological function, which is closely related to cell proliferation and metabolism. 15,16Table 6 indicated that Ca, Ma, Fe, Cu, Mn content were rich in the composite wine, while Pb, Cd was not detected.

Table 6
Trace elements content in banana and navel orange wine

CONCLUSIONS
Banana/orange composite wine with an alcohol concentration of 11.63% v/v were successfully prepared in the lab under the optimal condition of 28~30°C, 22% initial sugar content, 6% yeast, 6 days of fermentation.It was found that the sugar concentration had a critical effect on the alcohol fermentation.The order of impact factors on the ethanol fermentation were found to be: sugar concentration > fermentation time > fermentation temperature > inoculation amount.Sixteen compounds were identified by MS NIST in the banana/orange composite wine, which accounted for 96.15% of the total peak area, including 4 alcohols (79.43%), 11 ester (15.14%), 1 acid (1.58%).17 types of amino acids were identified in the composite fruit wine, 7 of which were essential amino acids that accounted for 21.51% of total amino acids, and 32.29% of which was proline.Ca, Ma, Fe, Cu, Mn were relatively rich in the composite wine, Pb was very low, and Cd was not detected.

Scheme 1 .
Scheme 1.The brewing process of banana and navel orange composite wines.

Fig. 1
Fig. 1 GC-MS total ion curent chromatogram of banana and navel orange wine component.

Table 1
Atomic absorption spectrometer working conditions

Table 3
Variance analysis of orthogonal experiment

Table 4
Analytical result of flavor components in banana and navel orange wine

Table 5
Analytical results of amino acid content in banana and navel orange wine