Yanan WANG, Songshan QIU, Rujin ZHOU, Cuicui JIANG

1. Guangdong University of Petrochemical Technology, Maoming 525000, China;

2. Jiangsu University of Science and Technology, Zhenjiang 212000, China

Fruit vinegar of physiology basicity has excellently superior function of health care to grain vinegar, and developing fruit vinegar doesn’t only save large numbers of grain, but also make the best of fruit resource and deal with unsalable problem of fruit.Orange vinegar,abundant in nutrition, contains quantities of organism acid as much as 10 kinds,and possesses of varieties of functions, such as balancing acid and base, preventing cancer, improving blood circulate, delaying the process of aging and resisting injection of fungi[1-2].Moreover,the research of orange vinegar contributes to change the laggard situation of processing orange (only processing 5%-8% of the whole orange). However, though some reports of developing orange vinegar, the present researches are limited in the level of simple fermentation process, the shortcoming existing in less factors considered and low yield of acetic acid and fermentation technology is in need to be improved further. Box-Behnken method has the capacity of obtaining high accuracy in short time[3-5], and response surface method can be used to optimize process and predict response value by multiple quadratic regression equation[6].The aims of this experiment were optimizing the technology of fermenting orange vinegar by Box-Behnken method, simultaneously exploring the action of single factor and interaction of double factors of inoculum size,original alcohol concentration and pH value.

Material and Method

Material and regent

Orange was purchased in Guan Du fruit wholesale market in Mao Ming, Guang Dong. Angel highly active dry yeast and Acetobacterium Balch were used for the fermentation.Sucrose, glucose and yeast extract were biochemical reagent,others were domestic analytic reagents.

Method

Activating and enlarging of active dry yeast Angel active dry yeast was taken into syrup of 2% at 35-40℃to rehydrate for 20 min, then taken into the electro-heating constant temperature cultivator (DHP-9162, Shen Zhen SAN LI Instrument) at 32 ℃to activate 2 h until quantities of bubble produced.

Enlarging cultivation of Acetobacterium Balch Active culture medium of Acetobacterium Balch was made up as the following compound ratio:yeast extraction of 1%,glucose of 1%,calcium carbonate of 1% and alcohol of 2%. In bacterial-free environment Acetobacterium balch wass inoculated in active culture medium, and cultivated in constant temperature cultivator for 24 h at 32 ℃for use.

Material treatment and enzymolysis of fermentation liquor Ripe and sound oranges were selected,scalded and squeezed into juice with a family juicer after removed peel and seeds.Orange juice was treated for 3 h in water bath of 45 ℃with enzymolysis of 1% pectase and 1% cellulase, filtered and put into triangular flask of 250 ml.

Alcohol fermentation of orange vinegar Orange juice was adjusted to sugar content of 12%-18% and pH value of 3.0-4.5 and sterilized for 30min at 65 ℃using pasteurization.Bacterial-free juice were added 3%-6% yeast active liquor into and kept at 30 ℃to ferment alcohol. The change of alcohol content was monitored every two days until alcohol content was steady.

Acetic acid fermentation of orange vinegar Enlarging culture fluid of Acetobacterium Balch was added into alcohol fluid to ferment acetic acid.The temperament was controlled at 30℃.The change of alcohol content was monitored every two days until alcohol content was steady.

Filter and sterilize of orange vinegar Orange vinegar was added diatomite into, stirred and placed still for 1-2 h until there was filter layer of 0.5-1.0 cm and no distinct turbidity. Then orange vinegar was filtered in vacuum condition and filter liquor was collected and sterilized for 30 min at 65 ℃using pasteurization.

Design of optimizing acetic acid fermentation by Box-Behnken method

After determining the preliminary range of the acetic acid fermentation variables through single-factor test and related references[9-10],a response surface design with three independent variables (X1,inoculum size;X2,original alcohol concentration; X3, pH value)at five levels was performed. The range of independent variables and their levels was presented in Table 1.

Measuring method

pH value was measured by a pH meter (PHS-3D type, Shang Hai Zuo Fei Lab Instrument Co.); Sugar concentration was measured by saccharimeter (RX-700α, Japan ATAGO Scientific Instrument Co.); Alcohol concentration was measured by potassium dichromate colorimetric method of related reference[7]; Acetic acid was measured by NaOH titration of related reference[8].

Result and Analysis

Single factor experiment of alcohol fermentation

Choice of original sugar content Sugar content is a very important factor in alcohol fermentation, which is indispensable for the growth and metabolism of Angel yeast and also the substrate of alcohol fermentation.Different original sugar contents of 12%,14%,16%,18% were shown in Fig.1 to impact the alcohol yield in following given conditions: inoculum size of 5%, temperature of 30 ℃and pH value of 3.5. Alcohol content was steadily increasing in early period,then the change of alcohol content was gradually decreasing in the following days until it was steady in 4-6thday.In 6thday the alcohol contents were measured of different fermentation conditions. The alcohol content reached 7.4%Vol when original sugar content was 16%, alcohol contents of fermentation fluid were 5.4%Vol, 6.3%Vol,7.2%Vol in original sugar content conditions 6 Aromyces cerevisiae, otherwise high sugar content will lead to the overgrowth of Saccharomyces cerevisiae and suppress alcohol fermentation. Given above, the sugar content of 16% was the most suitable for alcohol fermentation.

Choice of inoculum size Inoculum size is also a very important factor in alcohol fermentation. The effect of different inoculum sizes of 3%, 4%, 5%,6% was shown in Fig.2 on the alcohol yield in following conditions: sugar content of 16%, temperature of 30 ℃and pH value of 3.5. Alcohol content was steadily increasing in early period and came to the steady level gradually in 6thday,so the 6thday was treated as the terminal point of alcohol fermentation and in 6thday the alcohol contents were measured of different fermentation conditions. The alcohol content reached 7.3%Vol when inoculum size was 16%, alcohol contents of fermentation fluid were 5.7%Vol, 6.2%Vol,6.8%Vol in inoculum size conditions of 3%, 4%, 6%. It may be the reason that low inoculum size will limit the growth of Saccharomyces cerevisiae and high inoculum size will lead to the overgrowth of Saccharomyces cerevisiae and suppress alcohol fermentation. Therefore, inoculum size of 5% was the most suitable for alcohol fermentation.

Choice of pH The effect of different pH values of 3.0, 3.5, 4.0, 4.5 was shown in Fig.3 on the alcohol yield in following conditions: sugar content of 16%, temperature of 30 ℃and inoculum size of 5%. Alcohol content was firstly steadily increasing in early period,secondly the alcohol content was gradually increasing slowly and finally steady in the late period, in 6thday the alcohol contents were measured of different fermentation conditions.The alcohol content reached 7.3%Vol when pH value was 3.5, alcohol contents of fermentation fluid were 7.0%Vol,6.8%Vol, 6.2%Vol in inoculum size conditions of 3.0, 4.0, 4.5. The growth and fermentation of Saccharomyces cere-visia has different suitable pH,so the selection of pH have to consider growth and fermentation of Saccharomyces cerevisia. Therefore for balancing both, pH value of 3.5 was selected to be the most suitable for alcohol fermentation.

Table 2 The results of Box-Behnken experiments

Response surface experiment of acetic acid fermentation

The response surface experiment was used for the process optimization of acetic acid fermentation and the results were shown in Table 2.

Design-Expert soft was used for the regression analysis of results and presented the secondary multivariate regression equation:

Y=-184.330 00+16.230 21×X1+35.268 75 ×X2+42.030 833 ×X3-0.032 500×X1×X2-0.417 50×X1×X3-1.335 00 ×X2×X3-0.888 96 ×X12-2.560 83×X22-5.013 33×X32, in which response value was set as Y value(yield of acid,g/L)and variates were X1(inoculum size,%), X2(original sugar content,%)and X3(pH).

In the Table 3,Pmodel＜0.01 claimed the model extremely significant and Plackoffit=0.145 2＞0.05 turned out lack of fit was non-significant, in which lack of fit was the probability of non-fit between actual value and predicted value of model. R2adjwas 0.980 8, declaring 98.08% of response value change could be explained, R2was 0.993 1 and near to R2adj, declaring the degree of fit fine and low error. The whole model can be used to optimize fermentation process.

Analysis result of single factor action It was the relationship curve shown in Fig.4 that showed the effect of some single factor on the yield of acetic acid when setting other two factors. The yield of acetic acid firstly rose when enhancing inoculum size and original alcohol content, and then the yield started to decrease at arrival of the peaks, in which inoculum size had extremely significant effect (P ＜0.01)and original alcohol content presented significant effect (P ＜0.05). It was different that the yield decreased all the way with the growing of pH,and the effect was extremely signifi-cant(P＜0.01).

Table 3 Variance analysis for regression model

Analysis of interaction of response surface Fermentation pH was set in the level of 0. It was shown in Fig.5 that the increasing of original alcohol content made the yield of acid rise slowly firstly and then started to decline slowly at the peak when inoculum size set in the level of 0, and the current kept the same with the change of inoculum size,which was in a word the same as Fig.4B; the increasing of inoculum size made the yield of acid rise rapidly firstly and then started to decline rapidly at the peak when original alcohol content set in the level of 0,and the current kept the same as the change of original alcohol content,which was in a word the same as Fig.4A. Thus the interaction of inoculum size and original alcohol content was non-significant,which was corresponding to the non-significant interaction of X1X2in Table 3.

Original alcohol content was set in the level of 0. It was shown in Fig.6 that pH set in the level of 0, t he yield of acid rose firstly and then started to decline at the peak when increasing inoculum size, and the change kept the same when changing pH, which was in a word the same as Fig.4A;inoculum size set in the level of 0 and then changing, the improvement of pH both lowered the yield of acid rapidly, which was in a word the same as Fig.4C. Thus the interaction of inoculum size and pH was non-significant, which was corresponding to the non-significant interaction of X1X3in Table 3.

Inoculum size was set in the level of 0. It was shown in Fig.7 that pH set in the level of 0, the enhancement of original alcohol content made the yield of acid rose slowly firstly and then started to decline slowly at the peak,and the change of pH didn’t change the change current of the yield but did compel the curve to move to the direction of low original alcohol content.;original alcohol content set in the level of 0 and changing, the improvement of pH both lowered the yield of acid rapidly, which was in a word the same as Fig.4C. Thus the interaction of pH and original alcohol content was significant,which is corresponding to the significant interaction of X2X3in Table 3.

Identification of response surface model Design-Expert soft analyzed experiment results and ensured the optimal parameters. The optimal parameters were as follows: inoculum size of 8.52%, original alcohol content of 6.02% and pH of 3.31, and predicted yield was 53.095 3 g/L in the optimal fermentation condition. Test experiment was repeated 3 times according to the best fermentation process, the mean acetic acid yield was 53.11 g/L and relative error was low compared with predicted value,declaring response surface method advisable to optimize fermentation process parameters.

Conclusion

The research made sure the best fermentation process of orange vinegar.The optimal parameters of alcohol fermentation were as follows: original sugar content of 16%,inoculum size of 5% and pH value of 3.5; acetic acid fermentation was carried out in following optimal conditions: inoculum sizeof 8.52%, original alcohol content of 6.02% and pH value of 3.3, at last the yield was 53.11 g/L. The research built the regression equation. The regression equation was as follows:

The action of single factor and interaction of double factors on the yield of vinegar were shown as follows: the effect of inoculum size and pH were extremely significant (both P ＜0.01),that of original alcohol content was significant (P＜0.05);the interaction of inoculum size and original alcohol content was non-significant (P＞0.05), so was that of inoculum size and pH (P＞0.05), otherwise the interaction of original alcohol content and pH was significant(P＜0.05).

Acknowledgment

This research was financially supported in part by Guangdong Province Ministry of industry university research project (No. 2013B090600032) and Science and Technology Project Program of Guangdong Province (No.2014B040404027).

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