Biochemical Changes during Traditional Fermentation of Saudi Sorghum ( Sorghum bicolor L.) Cultivars Flour into Khamir (Local Gluten Free Bread)

found to protein and starch in vitro digestibility and antinutritional factors such as phytic acid, enzyme inhibitors and reported an increased in protein during natural fermentation of millet. Similarly, Cui et al. observed an increase in in protein content and most of amino acids especially lysine in maize after three days ’ fermentation. Abstract: Effect of traditional fermentation on pH, titratable acidity, proteins, amino acids, and sugars contents of three local sorghum cultivars namely Hamra, Biadah and Shahla used in making khamir local bread were investigated. During 24 fermentations, the pH of fermented dough dropped sharply and this was coincided with increase in total acidity. Fermentation was found to cause no significant change in protein content of the cultivars. Amino acid analysis, revealed slight insignificant improvement in lysine, and leucine content but there was a slight decrease in valine, phenyl alanine and arginine content in fermented dough. Glucose, fructose and maltose content of the three sorghum cultivars increased considerably in the early stages of fermentation, followed by decrease towards the end of fermentation. Low amount of sucrose detected in the three cultivars and it was completely utilized after 8 hrs. of fermentation.

pia, fin in India, Mahewu in Zimbabwe, Dolo and pito in west Africa and lahloh and Khamir in Saudi Arabia 6 . Fermentation process of sorghum flour had been reported to improve its nutritional quality and reduce antinutritional factors. Fermentation of sorghum for 36 or 24 hrs was also found to increase protein and starch in vitro digestibility and decrease antinutritional factors such as phytic acid, tannins, enzyme inhibitors 6,7 . Inyang and Zakary 8 reported an increased in protein during natural fermentation of millet. Similarly, Cui et al. 9 observed an increase in in protein content and most of amino acids especially lysine in maize after three days fermentation.
In Saudi Arabia, there are three sorghum cultivars Hamra, Bidaha and Shahla are grown in South-West region of Jazan, where it is traditionally utilized to prepare fermented traditional bread known as Khamir. The bread is prepared by mixing the sorghum flour with water, onion, lemon juice and fenugreek and fermented for 24 hr. The microbiological analysis of the micro-organisms involved in fermentation process of Khamir bread showed that lactic acid bacteria dominated over Enterobacteriaceae groups, yeasts and molds 10 . Fermentation process was also found to decrease antinutritional factors and increase protein digestibility 11,12 . However, there is no information is available regarding the biochemical changes occurring during traditional fermentation of sorghum in to Khamir bread. Therefore, the objectives of this study were to investigate the changes in pH, titerable acidity, proteins content, soluble sugars and amino acids content, occurring during traditional fermentation of sorghum cultivars mainly Hamra, Baidha and Shahla in to Khamir bread 2 Materials and Methods

Materials
The three sorghum varieties, Hamra, Baidha and Shahla used for this study were obtained from a local market in Jazan province South-west Saudi Arabia . The grain was manually cleaned from impurities and milled and the flour was then transferred to laboratory in Riyadh and stored at 25 until used.

Fermentation
Natural fermentation was carried out using the traditional process used by housewives as described by Osman 11 , briefly sorghum dough was prepared by mixing the flour with water and spices fenugreek, onion and lemon juice 1:0.8 ratio . The fermentation was carried out by adding 3 inocula from previous fermentation as a starter to the dough and incubated at 37 for 24 hours. The fermented dough is made into pancake shape with 2-3 cm thickness by hand and baked in clay or mud oven known as tann our for 15 min. Fermentation was performed in duplicate and sampled every 4 hr during fermentation period 24 hr . Samples were dried in a vacuum oven at 50 Heraucus LBS Co. , and milled to fine powder using a coffee miller then passed through a 60 mm mesh and kept at 4 in polyethylene bags for analysis.

Determination of pH and titratable acidity
The pH was monitored every 4 h interval using a digital pH-meter JenwayPH10 flested UK. . Titratable acidity was determined according to AOAC 13 . The titratable acidity was calculated as percent lactic acid.

Protein content
The crude protein of raw and fermented sorghum flour was determined by micro-Kjeldahl method as described by AOAC 13 .

Amino acid
Amino acids composition was determined following the AOAC 14 method. Using HPLC Shimadzu LC-10AD, Shi-madzu Corporation, Kyoto, Japan in duplicate samples after hydrolyzed with 6.0 N HCL at 100 for 24 hrs.

Determination of sugars
Glucose, sucrose, fructose and maltose content of raw and fermented sorghum flour were analyzed according to AOAC 13 , using HPLC Shimadzu LC from Shimadzu, Kyoto-Japan. Sugar standards were purchased from Sigma Sigma Chemical Co., St. Louis, Mo .

Statistical analysis
Data, expressed as mean and standard deviation, were statistically analyzed using one-way ANOVA Steel and Torri 15 . Duncan s multiple tests were used to compare means and significance was accepted at p 0.05.

Results and Discussion
3.1 Effect of fermentation on the pH and titrable acidity of sorghum cultivars The effect of fermentation on pH and titrable acidity of Hamra, Baidha and Shahla is presented in Figs. 1 and 2. In general, there was progressive decrease in pH with increase of fermentation time in the three sorghum cultivars. The pH of fermented dough of the three cultivars were was decreased and reached 4.43, 4.65 and 4.47 at the end of 24 hr fermentation for Hamra, Bidah and Shahla respectively. The pH dropped sharply at first from 8 h and then, there was slight insignificantly p 0.05 decrease up to 24 h in the three cultivars. The decrease in pH was concomitant with increase in titerable acidity. The titerable increased significantly p 0.05 from initial values 0.26, 0.27 and 2.0 to final values 1.37, 1.70 and 1.65 during 24 h fermentation for Hamra, Biadha and Shahla respectively. Similar observations were reported for several other cereals. Fadlalla et al. 16 observed that the pH of sorghum dropped from 6.05 to 3.79 and that of the sorghum supplemented with chickpea from 6.07 to 3.81 at the end of fermentation period 24 h . Similarly, Tou et al. 17 reported decrease in pH and increase titrable acidity during preparation ben-saalga, a traditional fermented millet-based gruel. According to Gassem 10 increase in titerable acidity at the end fermentation of sorghum dough in to Khamir bread is due to accumulation of lactic acid produced by increased number of lactic acid bacteria during fermentation. The final pH values of fermented sorghum cultivars dough mix were slightly higher than the safe inhibitory pH 3.6 to 4.1 for pathogenic bacteria. The high pH could be attributed to low lactic acid concentration due to shorter period of fermentation, the temperature or to the presence of other ingredients such as onion, lemon juice and fenugreek.in dough mix that may slow the fermentation process. Lactic acid is responsible for the desirable sour taste and the special flavor of the bread that are important for in utilization and acceptability.
3.2 Effect of fermentation on crude proteins of sorghum cultivars The change in protein contents of sorghum cultivars as function of fermentation time is shown in  20 also reported that the protein content of millet was not affected by fermentation. In same way, Yousif and El Tinay 21 and Ibrahim et al. 22 reported that the protein content of protein content of Dabar sorghum cultivar was fluctuated during the 24 h fermentation. However, prolonged fermentation time to 32 h slightly increase the protein content of sorghum dough. The slight insignificant reduction in protein content observed in this study could be attributed to the utilization of protein by microorganism for growth.  3.3 Effect of fermentation on Amino acids pro le of sorghum cultivars The effect of fermentation on amino acids content of Hamra, Biadha and Shahla sorghum cultivars is presented in Table 2. The amino acids composition of the unfermented sorghum flours showed that glutamic acids, leucine, alanine and aspartic acids were the major amino acids. The amino acids profile of the sorghum the three cultivars are in agreement with those reported by Abdel Rahman and Osman 23 . Fermentation process resulted in slight insignificant increase, leucine and lysine. Similarly, Fadlallah et al. 16 could not find any definite trend in amino acids during 24 h fermentation of sorghum and sorghum-chickpea blends. Cui et al. 9 , however, reported significant increase in most of the amino acids with largest increase in lysine, during 3 days fermentation of maize. Similarly, Hamad and Field 24 reported that natural fermentation of wheat, oats, sorghum and rice for 6 days significantly increased the essential amino acids, and available lysine content. On the contrary, Osman 25 , observed significant decrease in lysine, arginine and glycine after 24 h fermentation in millet dough. The slight change in amino acids content observed is probably due to short fermentation period used in this study.

Effect of fermentation on soluble sugars content of sorghum cultivars
The effect of fermentation on glucose, fructose, sucrose and maltose content of Hamra, Biadah and Shahla is presented in Fig. 3 A, B and C . The analysis of soluble sugars revealed that glucose is major soluble sugar in the three sorghum cultivars. The glucose content of unfermented Hamra, Biadha and Shahla dough was found to be 1.20, 1.58 and 1.12 gm/100 gm sample respectively. In the three   cultivars, the concentration of glucose increased sharply during early stages of fermentation and reached a maximum after 16 h and thereafter decreased. The increase of glucose was greater in Shahla 150.9 compared to Hamra 116. 7 and Bidah 110.2 . Similar to glucose, there was a sharp increase in fructose content in first 8 hrs of fermentation. It increased from 0.202 to 0.31, from 0.24 to 0.4 and from 0.2 to 0.36 for Hamra, Biadah and Shahla respectively. Increasing fermentation period up to 24 h markedly caused a decrease in fructose content to 0.172, 1.38 and 1.61 for the three cultivars. The result obtained here is in agreement with those of Osman et al. 19 who reported that the level of reducing sugars showed an increased and then a decreased as the fermentation time increases, during Kissra preparation. Similarly, Hamad and Field 24 observed increase and the decrease in reducing sugars during fermentation of wheat, rice, oats and sorghum for 6 days. The increase glucose content could be attributed to the action of microbial α and b amylase, sucrase and maltase, whereas the decrease at the end fermentation probably due to utilization of glucose as source of energy by microorganism.
Sucrose content was decreased rapidly and completely utilized after 8 h in the three cultivars. In contrast, the content of maltose increased sharply during the first 8 hr, it increased from 0.23 to 0.77, from 0.68 to 0.86 and from 0.51 to 0.80 for Hamra, Baidah and Shahla respectively. However, when the fermentation was continued to 24 h, the amount of maltose started to decrease at a steady rate and completely utilized at the end of fermentation period in both Biiadh and Shahla. Similar trend observed during fermentation of pearl millet to ben-salga, a fermented gruel 17 . The changes either as an increase or a decrease in maltose could be attributed to action of microorganism amylase that hydrolyzed starch to maltose and to the action of maltase associated with fermentation.
Therefore, in order to increase the protein and essential amino acid content, such as lysine the first limiting amino acids of fermented sorghum, the fermentation process should be carried out between 3-6 days or by using a fermenter with pure bacteria culture and controlled parameters for industrial application. The fermented sorghum flour could be utilized in formulation of weaning food, blend with wheat to produce cheap and nutritious bread, blended with local legumes for combating protein malnutrition in poor countries or help in solving major problem for celiac patient, due to the fact that the local bread is gluten free that has the poetical for commercial application.

Conclusion
Fermentation process of the three sorghum cultivars during preparation of the khamir gluten free bread resulted in decrease in pH, which was accompanied by increase in titerable acidity contents. The traditional fermentation process of sorghum cultivars was found to cause no significant change in protein. Amino acid analysis of fermented sorghum revealed slight insignificant increase in the content of some of the amino acids and decrease in others in three dough samples. Sugars study showed remarkable changes in soluble sugars content in the three sorghum varieties. These results indicated the need for longer fermentation time or addition of other process such as soaking or germination before fermentation to increase nutritional quality. The results indicate that fermentation for longer period would contribute in increasing protein and essential amino acids such as lysine the first limiting amino acid in cereal of the three local sorghum cultivars, that could be used in developing new cheap bakery products by mixing with wheat, in poor countries or weaning food for children in Asia and Africa by blending fermented sorghum flour with local legumes in order to combat protein malnutrition Kawakshior .