INTRODUCTION:

Microbial fermentation is form of energy-yielding metabolism in which organic substrate, usually a carbohydrate, is incompletely oxidized and organic carbohydrates acts as an electron acceptor¹. Majority of organic acids are produced during microbial fermentation². Fermented foods are those foods which have been subjected to the action of microorganisms or there related enzymes. Because of which desirable biochemical changes cause significant modification to food and its products.

This change is in related to taste, smell, consistency appearance and nutritional values of food³. Fermentative microbes majority produce different organic acids such as acetic and lactic acid which founds to inhibits the growth of other pathogenic bacteria. This is the reason that most of the fermented food serves with low pH benefiting preservation with nutritional assistances⁴.Phytic acid is commonly known as an antinutrient, storage form of phosphorus. Important mineral used in the production of energy and formation of structural elements⁵. Commonly phytic acid is found in grains, nuts and seeds. Which bounds strongly with available calcium, magnesium, zinc and iron from foods. Both interact each other in human/animal digestive tract and unabsorbed by body. Though phytic acid found to have some of the potential properties like antioxidant and cholesterol reducer^6,7,8. Some traditional food preparation methods like soaking, germination and fermentation found effective in reducing the amount phytic acid in foods/related products⁹. Regular fermented foods like idli, dosa, khaman, dhokla, locho, jalebi are quite popular in Gujarat region (India). These are not only fermented foods but also functional foods. Therefore consume by people as healthy food within the state and other parts of country. Inheritance of consuming sort of fermented food in selected region might be due to its geographical as well as climatic conditions. Cooking/making of fermented food is one of the ancient technique, practiced for thousands of years as potent tool for imparting longevity to foods/beverages. It is also known that fermentation process enhances the nutritional quality of foods by increasing the amount of vitamins and protein solubility. Imparting health beneficial valuess. Considering all above expects, an attempt was made in the present study to analyze some selected fermented foods for health beneficiary affects with relation to available minerals, fermenting microflora, phytic acid its anti nutritional mode and production of organic acid.

MATERIAL AND METHODS:

Fermented food(s) majority consumed in Surat (Gujarat) was used for present study. This includes dhokla, khaman, lo-cho, idli ,dosa and jalebi.

Sample collection:

Above mentioned fermented food were studied with respect to mineral analysis, fermentative micro flora and phytate value. Samples collected includes raw grain/beans/pulses/legumes, such as black gram (Phaseolus mungo), rice (Oriyza sativa), wheat (Triticumaestivam), bengal gram/cheakpea (Cicer arietinum), green gram (Vigna radiata).

Analytical methods:

The raw unfermented, fermented and processed food samples were analyzed for six different minerals needs for effective functioning of human body. This includes calcium, magnesium, zinc, iron, sodium and potassium.

Calcium and magnesium analysis:

Preparation of sample for analysis:

5 g of food sample was weighed into crucible to make it dry-ash. This was allowed by heating at 100-250ºC in hot air oven for 2-4 hrs. Available sample was allowed to dissolve in 10 ml concentrated HCl solution. Further boiling and evaporation was done to make sample dry. Available residue was re-dissolved in about 20ml 2N HCl, with mild boiling. In last filtered and final volume make up to 100 ml.

Ca and Mg analysis:

Titrimetric method was designed to analyse calcium and magnesium. Were 5 ml of sample prepared as above was mix with 5 ml of ammonium buffer and diluted up to 100 ml with de-ionized water. In to which small pinch of erichrome black T was added and solution was allowed to heat up to slight boiling. After titrated against EDTA solution until the dark pink/reddish color changes to blue. The end point noted as ‘A’. The amount of calcium in sample (mg/L) was calculated as follows,

Calcium (mg/L) = F × B × 1000 / Volume of sample

Where, B is ml of consumption of EDTA by Ca alone and factor value for calcium (F) is 2. Afterword another 5 ml sample was collected and to which 5 ml of NaOH solution was added. Dilution was allowed to make 100 ml with deionized water. Pinch of murexide was added as indicator. In last titrated against EDTA until the pink color turns to blue. End point was noted as ‘B’. The amount of magnesium in sample (mg/L) was calculated as follows

Magnesium (mg/L) = F × (A – B) × 1000 / Volume of sample

Where, A is ml of consumption of EDTA by Ca and Mg while B is ml of consumption of EDTA by Ca alone and factor value for magnesium (F) is 1.2¹⁰.

Zinc analysis:

Sample for Zn analysis was made in similar manner as shown above for that of Ca and Mg. Followed by dilution to 250 ml with deionized water. In next step 25 ml of aliquots collected each into three different sets. To which 15 ml of deionized water, 9 ml ammonium buffer of pH 10 was mixed. In last 2-3 drops of eriochrome black T added prior to titration. Prepared sample was then titrated with standardized EDTA solution until the pink solution turns light blue. About 25 ml of aliquot was titrated out of 250 ml total volume (each aliquot representing one- tenth of the total sample) mg of zinc in the sample was calculated as follows¹¹.

mmol_Zn= mmol_EDTA= mmol_EDTAxV_EDTA=mmol_EDTAxVep

The mass of Zn obtained in a single titration in mg, is equal to the number of mmol of Zn 10 times its molar mass (MM):

mg_Zn= mmol_Zn x MM_Zn= mmol_Zn x 65.38mg/mmol

Iron analysis:

For amount of iron analysis sample preparation was done by flame heating. Were 2.5 g of food sample allowed to turned ash. After cooling 10 ml of 2.0 M HCl added with continues stirring for 1-3min. In last 10 ml of distilled water added. To the filtered sample 2.5 ml of 0.1 M potassium thiocyanate was added. Absorbance was read at wavelength of 458 nm using UV-VIS spectrophotometer, unknown samples compared by help of standard graph¹².

Sodium and potassium analysis:

For sodium and potassium analysis flame photometry technique was used (Systronics Type 130 flame photometer). Samples were analyzed in triplicate sets in di-acid mixture (4 parts HNO₃and 1 part HClO₄) according to the procedure of Johnson and Ulrich. For digestion, one gram of each food sample was mixed with 20 ml of di-acid mixture and kept overnight by covering it to avoid any contamination. Sample digested at low temperature using hot plate. Digestion was allowed to continue till the liquid turns into colorless. In last sample dilution make to 100 ml using double distilled water¹³.

Phytate analysis:

Amount of available phytic acid was determined by complexometric titration method¹⁴ as modified by Garcia Estepa¹⁵.Were 5 g of food sample was extracted under continues agitation with 40 ml of extraction solution composing 10g/100g Na₂SO₄ in 0.4 M/L HCl for 3 hrs at room temperature. Which was then centrifuged at 5000 rpm for 30 min. Collected 10 ml of supernatant was mixed with 10 ml of 0.4 M/L HCl, 10 ml 0.02 M/L FeCl₃ and 10 ml 20 g/100 g sulphosalicylic acid. Mixture was allowed to boiled in water bath for 15 min and again centrifuged for 10 min at 5000 rpm. In last filtration was done and residue allowed to washed 3-4 times with distilled water. Supernatant of the filtrate was diluted to 100 ml from dilution 20 ml of aliquot was collected and adjusted to pH 2.5 with 0.5 M glycine which was further diluted to 200 ml in last heated at 70-80º C. After cooling titrated with 50 mM/L EDTA solution till color changes from red/maroon to clear yellow. Phytic acid content was calculated using Fe: P (4:6) ratio¹⁵. Following formula was used for calculation of phytic acid percentage.

% Phytic acid =0.66 (10-v) /m

Where, v = volume of EDTA (ml) and m = sample mass (gram).

Microbial analysis of food samples:

Majority fermented food contains probiotic commonly occurring microbial flora which are non pathogenic and responsible for fermentation with production of organic acids. For isolation fermented batter of following food were collected from households into sterile containers. Which includesidli, dosakhaman, lo-cho, dhokla and jalebi. Added 10% (w/v) sample were homogenized in phosphate buffer (0.1M pH 7.2). Serially dilution was allowed for isolation of fermentative bacteria different lab media like man rogosasharpe agar, tryptone glucose yeast extract agar, malt extract- yeast extract peptone glucose agar and Streptococcus lactis differential agar base medium. Incubation period was 37˚c for 24-48 hrs.

Lactic acid Estimation:

Pure culture of isolates from selected food samples were spot inoculated on the Luria-Bertani agar medium containing 0.4% CaCo₃ and 1% glucose with incubation of 37˚C for 48 hrs. The lactic acid production was determined by having clear zone around microbial growth¹⁶. Positive isolates were further selected for lactic acid production in liquid broth. By inoculating pure culture into luriabertani broth containing 0.4% CaCo₃and 1% glucose by incubation at 37˚C for 48 hrs on rotary shaker at 150 rpm/per min. After centrifugation percent lactic acid produced in liquid media was determine by collecting 10 ml of fermented broth free from cell debris. Indicator phenolphthalein was used and titrated with 0.1 M NaOH for the appearance of pink colour. Titrableacidity was calculated as lactic acid % W/V. Each ml of 1 N NaOH equivalent to 90.08 mg of lactic acid. The titrable acidity was then calculated using following formula¹⁷ .

(0.1MNaOH) X Vol. of NaOH (in liter) X 90.08* X100

Lactic acid % = ------------------------------------------------------------------

Weight of sample

*90.08 g/mol is the molecular weight of lactate.

RESULTS AND DISCUSSION:

Fermented foods include varying method of processing and cooking. Some examples of selected fermented foods being consume daily by local people as source of breakfast or as part of lunch/dinner is shown below in table no.1.

Table 2 Biochemical characterization for food fermenting microbes

Biochemical test	Isolates
Biochemical test	I-3	I-4	I-6	I-7	K-6	K-4	J-17	J-18	J-19	D-4	D-9	K-8	K-1
H₂S production test	-	-	-	-	-	-	-	-	-	-	-	-	-
Nitrate reduction test	-	-	-	-	-	-	-	-	-	-	-	-	-
Catalase test	-	-	-	-	-	-	-	-	-	-	-	-	-
Gelatin hydrolysis test	-	-	-	-	-	-	-	-	-	-	-	-	+
Starch hydrolysis test	-	-	-	-	-	-	-	-	-	-	-	-	-
NaCl tolerance test
1%	+	+	+	+	+	+	-	-	-	-	+	+	+
3%	+	+	+	+	+	+	-	-	-	-	+	+	+
5%	+	+	+	+	+	+	-	-	-	-	+	+	+
Phenol tolerance test
0.1%	+	+	+	+	+	+	-	-	-	-	+	+	+
0.2%	-	-	+	-	-	-	-	-	-	-	+	-	-
0.4%	-	-	-	-	-	-	-	-	-	-	-	-	-
Milk fermentation	+	+	+	+	+	+	+	+	+	+	+	+	+

^a(+)Positive test :(-) Negative test

Table no 3 Microbial sugar fermentation test for selected isolates

Sugar fermentation test

Isolates

I-3

I-4

I-6

I-7

K-6

K-4

J-17

J-18

J-19

D-4

K-8

K-1

Glucose

A,G

Sucrose

Lactose

A.G

Maltose

Mannitol

A,G

Xylose

Fructose

Mannose

Cellobiose

Mellibiose

Raffinose

Arabinose

^aA= Acid, Al= Alkaline, G= Gas production

The contemporary study on mineral analysis of above mentioned fermented foods reveals that the mineral contents of unfermented fermented and processed food varies. Analyzed mineral values are significantly different for all. As per different research outcomes and literature survey it was found that fermentation and food processing/cooking affects the functional properties of available minerals. Cereals, pulses and beans are important substrates for fermented foods and are staple in India subcontinent¹⁸. Preponderance plant based diets are often associated with micronutrient deficits, exacerbated in part by poor micronutrient availability. Evident that the absorption ability of Ca, Mg, Fe, K and Zn majority affected. The interactions between nutrients and antinutritional factor¹⁹. All these factors have more influence on bioavailability of the micronutrients in plant related foods. Interference of antinutritional biochemicals is found with digestion, thereby preventing utilization of competent related to proteins and minerals²⁰.

It was also noticed that many times they are capable of precipitating harmful effects in human, with obvious toxicity ranging from severe reduction in food consumption and nutrient utilization to profound neurological effects²¹. Plant based diet normally contains considerable amount of phytate reducing dietary minerals²².

Minerals form an integral part of functionally important organic compounds²³. They are very essential for the normal functioning of muscles, heart, nerves and in the maintenance of body fluid²⁴. Due to the world wide pervasiveness of phytate and its divergent effects on health, there has been collective attention in the development of practical approaches to reduce phytate content of essential food materials. Traditional methods of food processing such as soaking, germination and fermentation activates phytase which hydrolyses/degrades phytate into lower inositol phosphate have been reported to significantly reduce phytic acid. In present study commonly consumed, fermented foods prepared and consume generally in Surat region (Gujarat) and other parts of India was subjected to expand the In Vitro mineral availability.

All the fermented and processed food samples reconnoitered had relatively lower levels of phytate as compared with the unfermented or raw samples. Similar results were reported by others²⁵. The phytate contents of unfermented dhokla batter was 8.48 ± 0.01 mg/100 g which was reduced to 1.412 mg/100 g in fermented batter, further the content reduced to 1.112 mg/100 g in processed product. Marfo(1990) also reported reduction in phytate content during fermentation of bread and other wheat products²⁶. Fermentation increased the activity of available natural phytase enzyme leading to more phytate degradation²⁷. Similar results were reported by Oboh²⁸ in fermented cassava products.

The reduction in phytic acid content after processing was also significant. Current research on selected fermented samples resulted in a decrease in phytic acid content of the products with increase in the Ca, Mg, Zn, Fe, Na, K contents. The removal of antinutritional factor by fermentation technology improves the nutritional value of foods. Different food processing methods similar as steaming/heating/frying resulted in better availability of Ca, Mg Zn, Fe, Na and K. Also phytate to mineral ratio of fermented and processed product are lower that of the raw foods. Thus minerals are more available from products than from the raw foods. Therefore, food fermentation as being a simple and inexpensive method must promoted at the household to large level to reduce the amount of nutrient inhibitors like phytate for improving the availability of mineral from the diet.It is known that pathogenic microorganisms normally found in food will not be able to grow in an acid environment, which is at pH below 4. This acidity is normally found in fermented foods majority by lactic acid. Attempt was made to study production of lactic acid in selected fermented foods. At the starting before fermentation, food is vulnerable to microbial or other type of contamination since it does not have any acidity. A study mainly shows that acid environment suppresses pathogenic bacteria as pathogenic species were not isolated from selected samples. With intent to check lactic acid production particular isolates were allowed to undergo microbial fermentation using selected media. Production of lactic acid was 0.16%, 0.17%, 0.18%, 0.18%, 0.18%, 0.22%, 0.22% and 0.28% for isolates coded as K-8, I-7, K-4, D-9, J-17, D-11, J-18 and I-6 respectively. Noteworthy the total amount of lactic acid at the end of the fermentation was found to increase. Such acidity may be due to other organic acids produce by food fermenting flora. Lactic acid was determined qualitatively by plate assay methods. Lactic acid production increases as the fermentation proceeds. There may be difference in buffering capacity for the different raw materials. Heating and cooking reduces acidity to some extent. The production rate of lactic acid was also influenced by the amount of inoculum. (butter milk/curd added before fermentation). Majority value of pH range of fermented materials changes in relation with different raw materials used. Almost a strong relation of pH development to the growth of Lactobacilli found in all cases.

However, the growth of Lactobacilli began to be stable when pH dropped to 4². This was found true in our research study were recorded acidic pH values at the end of fermentation by isolating Lactobacillus flora. Calcium content of unfermented batter of jalebi was recorded as 28 mg/100g which was lowest Ca value compared to other were the khaman and lo-cho samples shows highest value of about 36 mg/100g. Such kinds of results found to similar with other minerals like Mg, Zn and Fe. Some variable findings were also noticed, in this case amount of available Na decreases in all food samples as fermentation processed and cooking was done. In case of available K it was recorded that amount of potassium decreases after fermentation in all food samples, but slightly increase was measured after cooking/processing for khaman, locho and jalebi. This may be due to phytic effect and processing method.

CONCLUSION:

Fermentation and processing resulted in decrease level of phytic acid content in all selected food samples. Calcium, magnesium, zinc and iron content are increased after fermentation whereas sodium and potassium contents decreased. Thus, indicating that the calcium, magnesium, zinc, and iron are more accessible in fermented foods compared to raw foods. The results also showed that lactic acid bacteria have the potential to produce the lactic acid at the optimized fermentation condition reducing the level of nutrient inhibitors like phytate with improving existing mineral values.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 19.05.2017 Modified on 14.06.2017

Asian J. Research Chem. 2017; 10(4):531-540.

DOI:10.5958/0974-4150.2017.00088.8

Sample no.	Product	Ingredients	Fermentation Process	Food Processing/Cooking
1	Idli and Dosa	Rice Black gram Salt Buttermilk	Soaking overnight, followed by making paste and mixing with buttermilk to proceed fermentation for about 24-48hrs	Idli basically steamed and dosa hot dry to semi fried
2	Dhokla	Chickpea flour Green gram Buttermilk	Mixing flour of all ingredients with butter milk and fermentation for 24-48 hrs.	Dhokla are generally steamed
3	Khamanand Lo-cho	Chickpea flour Black gram Buttermilk	Soaking overnight and mixing paste with butter milk to allow fermentation for 24-48 hrs.	Khamanavailable as steamed food Lochoas steamed food with hot dry to semi fried
4	Jalebi	Chickpea flour MaidaCurd	Curd, and water mixed with maida to make a thin batter with fermentation process of 24-48 hrs.	Deep fried and soaked in sugar syrup