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. 2021 Mar 30;58(9):3589–3597. doi: 10.1007/s13197-021-05088-3

Reaction kinetics of physico-chemical attributes in coconut inflorescence sap during fermentation

R Pandiselvam 1,✉, M R Manikantan 1,✉, Shalu M Binu 1, S V Ramesh 1, Shameena Beegum 1, Murali Gopal 2, K B Hebbar 1, A C Mathew 1, Anjineyulu Kothakota 3, R Kaavya 4,5, Sandip Shil 6
PMCID: PMC8292541  PMID: 34366476

Abstract

The study on fermentation kinetics of the coconut inflorescence sap is important to understand its shelf life at different storage conditions and to develop suitable value added products. The coconut inflorescence sap collected by using in-house developed coco-sap chiller device is called Kalparasa. The fermentation characteristics of Kalparasa were investigated at every 1-h interval under ambient (31 ± 2 °C) and refrigerated (5 ± 1 °C) storage conditions. The results reveal that pH of the sap and total sugar content decline rapidly under ambient conditions than under refrigerated conditions. Acidity, turbidity, and reducing sugar content significantly (p < 0.001) increases for the sap stored under ambient conditions. The reaction rate constant (k) of the vitamin C and total sugar degradation increases with the atmospheric fermentation. The degradation kinetics of vitamin C and total sugar in Kalparasa during natural fermentation (ambient condition) follow second-order equation whereas the reducing sugar follows the first-order equation.

Keywords: Coconut inflorescence sap, Neera, Kalparasa, Fermentation, Palm sap, Reaction kinetics

Introduction

Coconut (Cocos nucifera L.) is an important economic crop of India and other tropic and sub-tropic regions. Traditionally, coconut palm sap or neera has been tapped using an earthen pot coated with lime and the sap obtained had the characteristics of oyster-white color, translucent appearance, and sweet taste (Gupta et al. 1980). Scientific reports reveal that the neera is a highly nutritious drink and an excellent digestive agent. The palm sap collected using earthen pot has various constituents: 14.1 mg/100 g of total phenols, 350 mg/100 g of free amino acids, 6.52 g/100 g of total sugars, and 4.73 mg/100 g of vitamin C (Hebbar et al. 2018).

The traditional practice of neera tapping prevents the fermentation albeit only partially. Thus, ICAR- Central Plantation Crops Research Institute (CPCRI) has developed the device “coco-sap chiller” to tap fresh, hygienic, nutritious [total phenols (21.9 mg/100 g), free amino acids (901 mg/100 g), total sugars (15.96 g/100 g), and vitamin C (13.45 mg/100 g)] and unfermented sap (zero alcoholics) from the coconut palm (Hebbar et al. 2015). Coconut sap collected using the coco-sap chiller is called as Kalparasa and registered under the trademark of class 32 (non-alcoholic drinks). Kalparasa contain 1.5, 1.8, 2.5, and 4.6 folds high total phenols, antioxidants, amino acids, and flavonoids, respectively, than traditionally obtained neera. Sugar and honey (concentrated sap) (Hebbar et al. 2013), vinegar (Shameena Beegum et al. 2018), alcoholic beverages and biofuel (Xia et al. 2011) are the major value-added products prepared from neera. Further, the sugar produced from Kalparasa was found to be rich in polyphenols, amino acids, antioxidants, vitamins, and flavonoids (Hebbar et al. 2015).

Kalparasa is highly susceptible to natural fermentation due to its high sugar content (10–15%) and neutral pH. This optimal nutrient conditions of Kalparasa enhance the growth of native microflora (lactic acid bacteria) and yeasts (Saccharomyces cerevisiae) resulting in alcoholic fermentation (gets converted to toddy and the alcohol content increases up to 5.8%) and then acidic fermentation (4–7% acetic acid) (Iwuoha and Eke 1996). Kalparasa is nutritious when it tapped fresh but emanates very harsh flavor during fermentation and becomes unpalatable. The fermentation process is rapid under sunlight conditions. Thus, the collection and storage environment plays a vital role in maintaining the nutrient composition of the sap.

The physico-chemical properties and flavor profile of fresh and fermented coconut inflorescence sap which is collected by traditional earthen pot has been reported (Atputharajah et al. 1986; Ysidor et al. 2015; Shetty et al. 2017; Ramalakshmi et al. 2018). The reports available are representation of quality profile of partially fermented white color coconut inflorescence sap, which is collected by using traditional earthen pot. However, the quality profile changes of pure unfermented golden brown color coconut inflorescence sap (Kalparasa), which is collected using coco-sap chiller device under ambient and refrigerated conditions are not available. Even though, Kalparasa is a rich source of sugars, minerals, and vitamins, the inconsistency in the nutritional profile due to the rapid fermentation has severely dented its market potential. Also, biochemical profiling of reducing sugars, total sugars, turbidity, and vitamin C in the sap is vital to produce better end products (vinegar, biofuel, etc.,) from Kalparasa.

The importance of the reaction kinetics of the bioactive components in fruit juices during fermentation is better illustrated by the utility of kinetic equations to predict the quality changes. First-order kinetics is generally used to describe the degradation pattern of food bioactive components (Pandiselvam et al. 2017a). The fermentation kinetics of the Kalparasa mainly depends on the method of sap collection and environmental conditions. Hence, this research work aims to investigate the physico-chemical changes in Kalparasa during natural fermentation under ambient and refrigerated conditions and to decode the kinetics of physico-chemical attributes in Kalparasa using the mathematical models.

Materials and methods

Kalparasa collection

Kalparasa was collected from the coconut (Cocos nucifera L.) farm orchard of ICAR-Central Plantation Crops Research Institute, Kasaragod, using the insulated “coco-sap chiller” device specifically designed for the purpose. The dimensions and construction details of the coco-sap chiller were described in our previous work (Hebbar et al. 2018). Kalparasa was tapped from the unopened spadix of the healthy coconut palm from 6 PM to 6 AM. The ice crystals present in the coco-sap chiller maintain the temperature of the box at 4 ± 2 °C for 24 h. This low-temperature condition reduces the activity of microbes including yeasts during the long process of tapping. Also, the Kalparasa collected was free of dust, insects, ants, and pollen. Kalparasa collected in the polyethylene bag (100-micron thick low-density polyethylene film) was immediately transported to Agro-Processing Complex, ICAR-CPCRI using an icebox (< 5 °C) to prevent any fermentation during the transit and the samples were stored at − 18 °C until further use.

Experimental conditions

Fresh Kalparasa stored under room temperature (31 ± 2 °C) and refrigerated temperature (5 ± 1 °C) in 500 ml polypropylene bottles. The fresh Kalparasa are available in the market from 10 AM to 4 PM (6 h) and hence the Kalparasa samples were analyzed for different physico-chemical parameters (pH, total solids, titrable acidity, turbidity, vitamin C, total sugar, and reducing sugar) at an hourly interval up to 6 h.

Analysis of physico-chemical parameters

pH, TSS, and turbidity of the fresh Kalparasa were analyzed by using digital pH meter (Thermo Scientific Eutech pH 150; Resolution: 0.01 pH; Accuracy: ± 0.01 pH), pocket refractometer (Company: Atago Ltd., Tokyo, Japan; Resolution: Brix 0.1%; Accuracy: ± 0.2%), and turbidity meter (Oakton T-100 Turbidity Meter; model: AO-35635-05; Range: 0.01 to 1000 NTU) respectively, at every 1-h interval. The titrable acidity of a Kalparasa was measured by reacting with the acids and a base [sodium hydroxide (NaOH)]. The endpoint was measured by an acid-sensitive colour indicator (phenolphthalein) and expressed as percent acetic acid. Total sugar and reducing sugar were determined following the procedure of the phenol–sulfuric acid method (Dubois et al. 1956) and Nelson and Somogyi method (Somogyi 1952), respectively. Vitamin C was quantified as per AOAC (2000).

Reaction kinetics

The reaction kinetics was studied to predict the effect of fermentation time and storage condition on quality attributes (Pandiselvam et al. 2015). Reaction order, half-life, and rate constant (k) play an important role in predicting food quality loss during the fermentation. The zero-order (Eq. (1)), first-order (Eq. (2)), and second-order (Eq. (3)) equations were used to understand the behavior of physico-chemical quality changes during fermentation.

Ct=Co-kt 1
ln(Ct/Co)=-kt 2
1Ct-1Co=kt 3

where, Ct and Co indicate the physico-chemical parameters of the Kalparasa at time t and 0, respectively, t is the fermentation time (h) and k is the rate constant (h−1).

The stability of the degrading components such as vitamin C and total sugars in the Kalparasa during fermentation was calculated via half-life (t1/2) value, which indicates the time required for a fifty percent reduction in the original value at the same temperature. The t1/2 was calculated using Eq. 4 (for zero-order), 5 (for first-order), and 6 (for second-order) as follows:

t1/2=12k[Co] 4
t1/2=0.693k 5
t1/2=1k[Co] 6

Microbial analysis

For determining the microbial contents in freshly collected Kalparasa kept in ambient and refrigerated conditions, an hourly time-course study was conducted until 6 h period. Duplicate samples from each condition were serially diluted and plated on Nutrient Agar medium for enumerating general heterotrophic bacterial population and on Sabouraud’s Agar medium for enumerating yeasts and molds. Plate replication was done in triplicate for each sample. Plated samples were kept in BOD at 28 ± 2 °C. Observation of colony growth was made by 24 to 28 h for bacterial, yeasts and mold growth. The results were furnished as cfu/ml sample.

Statistical analysis

All the experiments were replicated thrice and the mean ± standard deviation is reported. The individual and combined effect of parameters (fermentation time and storage condition) on physico-chemical properties of Kalparasa were analyzed by factorial CRD design. Statistical software AGRES (Ver. 7.01) was used to analyze the statistical significance between the samples. The kinetics model parameters were estimated using R software (Ver. 3.6.0).

Results and discussion

Changes in pH and acidity during fermentation

Changes in pH and acidity of Kalparasa during the course of natural fermentation under atmospheric and refrigerated storage conditions are presented in Table 1. The pH of Kalparasa stored under atmospheric conditions rapidly decreased from 6.77 to 3.74 during the storage period (6 h). Hebbar et al. (2015) reported that fresh Kalparasa has a pH value of 7.5, however, it depends on the geographical locations, environmental conditions, and genotypes (Ghosh et al. 2018). Kalparasa is not suitable for consumption if the pH decreases to 6. But the Kalparasa stored under refrigerated condition (5 ± 1 °C) maintained its original pH beyond 6 h of storage. The atmospheric storage temperature (31 °C), could have enhanced the fermentation rate. In the first 3 h of atmospheric storage, the pH of Kalparasa was reduced to 5.87 from the original pH of 6.77. It was followed by a drastic decrease in pH from 5.87 to 3.74 at the end of 6 h. The completely fermented coconut inflorescence sap has a pH value of 3.3 to 3.5 (Hebbar et al. 2015; Ramalakshmi et al. 2018). This could be attributed to the production of acids during the fermentation. Titratable acidity value has significantly increased from 0.02 to 0.28% acetic acid (1300% increase) during the course of storage in atmospheric conditions. Fresh Kalparasa undergoes lactic acid fermentation followed by alcoholic fermentation and then final acetic acid fermentation when it is exposed to the atmosphere condition (Xia et al. 2011). Lactic acid production is high under enhanced sugar condition and controlled pH (6.2) in fermented sweet sorghum juice (Wang et al. 2016). The increase in titratable acidity (% acetic acid) of the Kalparasa shows that the increase in acid production is due to the action of bacteria and yeasts. Similar results were reported by Ysidor et al. (2015) and Ramalakshmi et al. (2018) for coconut inflorescence sap, wherein the titratable acidity value increased from 0.26 to 1.85 g. (100 g)−1 (612% increase) and 15 to 177 mg L−1 (1080% increase) during the storage of 120 h and 40 h periods, respectively. The effect of fermentation temperature and duration of fermentation and their interactions (fermentation temperature × time) have a significant effect (p < 0.001) on the pH and titratable acidity of the sap (Table 2).

Table 1.

Changes in physico-chemical characteristics of Kalparasa during fermentation

Storage time, h Storage condition pH TSS, °Brix Turbidity, NTU Acidity, % acetic acid Vitamin C mg/100 g Total sugar, % Reducing sugar, %
0 Fresh Kalparasa 6.77 ± 0.03 15.70 ± 0.26 26.99 ± 0.83 0.02 ± 0.00 14.94 ± 2.13 13.17 ± 1.26 0.27 ± 0.02
1 Atmosphere 6.55 ± 0.02 15.40 ± 0.36 28.15 ± 1.21 0.02 ± 0.00 14.62 ± 1.16 12.53 ± 1.14 0.39 ± 0.03
Refrigerated 6.69 ± 0.04 15.53 ± 0.21 27.20 ± 1.66 0.02 ± 0.00 14.84 ± 0.36 12.67 ± 0.30 0.33 ± 0.03
2 Atmosphere 6.36 ± 0.06 15.23 ± 0.15 42.56 ± 8.50 0.02 ± 0.01 14.56 ± 0.72 11.84 ± 0.49 0.40 ± 0.01
Refrigerated 6.66 ± 0.02 15.33 ± 0.37 28.90 ± 0.60 0.02 ± 0.00 14.60 ± 0.33 12.44 ± 0.90 0.32 ± 0.03
3 Atmosphere 5.87 ± 0.12 15.12 ± 0.43 88.73 ± 20.52 0.03 ± 0.01 14.19 ± 0.33 11.73 ± 0.18 0.54 ± 0.04
Refrigerated 6.67 ± 0.07 15.03 ± 1.25 30.62 ± 2.46 0.02 ± 0.01 14.60 ± 0.16 12.28 ± 0.96 0.33 ± 0.01
4 Atmosphere 4.46 ± 0.15 15.06 ± 0.15 422.66 ± 49.94 0.13 ± 0.01 13.65 ± 0.69 10.81 ± 0.37 0.73 ± 0.05
Refrigerated 6.59 ± 0.08 15.14 ± 0.22 32.29 ± 1.60 0.03 ± 0.01 14.47 ± 0.77 12.24 ± 0.38 0.35 ± 0.02
5 Atmosphere 3.93 ± 0.06 15.10 ± 0.26 584.88 ± 53.92 0.23 ± 0.07 11.64 ± 0.29 10.50 ± 0.35 0.84 ± 0.09
Refrigerated 6.59 ± 0.01 15.15 ± 0.43 35.61 ± 2.18 0.03 ± 0.01 14.45 ± 0.81 12.05 ± 1.10 0.37 ± 0.03
6 Atmosphere 3.74 ± 0.01 14.87 ± 0.05 652.33 ± 25.76 0.28 ± 0.05 11.20 ± 0.29 9.75 ± 0.49 0.93 ± 0.05
Refrigerated 6.57 ± 0.03 15.03 ± 0.20 37.37 ± 3.34 0.05 ± 0.01 14.21 ± 0.58 12.02 ± 0.83 0.38 ± 0.03
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Atmosphere, atmospheric storage (31 ± 2 °C), refrigerated, refrigerated storage (5 ± 1 °C)

Table 2.

ANOVA for physico-chemical characteristics of Kalparasa during fermentation

Parameters df pH TSS Turbidity Titratable acidity Vitamin C Total sugar Reducing sugar
Storage atmosphere (A) 1 4909.066*** 9.193** 1713.830*** 103.594*** 0.075NS 0.087NS 385.620***
Storage time (T) 6 759.524*** 1.420NS 365.204*** 34.296*** 10.779*** 12.687*** 52.119***
A*T 6 692.986*** 0.755NS 345.590*** 24.050*** 3.780** 1.211NS 34.323***
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*** significant at 0.001, ** significant at 0.01, * significant at 0.05, NS non significant

Changes in total soluble solids and turbidity during fermentation

One gram of sucrose in 100 g of Kalparasa represents one degree Brix. Santos et al. (2013) contradicted that the °Brix value is not necessarily based on sugars content. Total soluble solid (TSS) of Kalparasa was found to reduce by 5.29% (from 15.70 to 14.87 °Brix) during the course of atmospheric fermentation. The conversion of sugars into alcohol due to the action of bacteria and yeast causes the reduction of °Brix. A slight increase in °Brix value was observed during the 5th hour of fermentation. This may be due to an increase in hydrogen (H2) production by the action of microbes (Ngoc et al. 2013). Research reports have highlighted that the final alcohol content will depend on the initial °Brix value. Santos et al. (2013) reported that the initial sugar levels (16°Brix) would yield approximately 8% (v/v) alcohol after fermentation. Similarly, the coconut inflorescence sap yields an alcohol content of 5.17% (Ramalakshmi et al. 2018) and 4.1% (Shetty et al. 2017) at the 40th and 13th hour of natural fermentation, respectively.

The turbidity of Kalparasa stored in atmospheric conditions rapidly increased from 26.99 NTU to 652.33 NTU (2316% increase) at the 6th hour of storage. It could be due to the disintegration of the particles during fermentation. The analysis of variance (ANOVA) indicated that the fermentation temperature and duration of fermentation and their interactions have a significant effect (p < 0.001) on turbidity (Table 2). During the bioconversion of sucrose to monosaccharides (glucose and fructose) by lactic acid bacteria number of molecules tend to increase (Wang et al. 2016). The initial golden brown color of the Kalparasa (0th hour of storage) changes to milky white at 6th hour of atmospheric storage (Fig. 1). This also causes an increase in turbidity value. Kaya and Unluturk (2016) reported that changes in the turbidity level of grape juice could be attributed to the change in color pigments. The non-significant difference in turbidly was noted in Kalparasa stored under refrigerated conditions. This could be attributed to the inhibition of fermentation rate in the refrigerated temperature (5 ± 1 °C). The foul odour and slightly whitish color were observed after 2 h of fermentation in atmospheric conditions. This may be due to the growth of contaminated microbes (Peerajan et al. 2016). Phenolic compounds are contributing to the antioxidant properties and color of Kalparasa (Hernandez et al. 2007). Atmospheric fermentation enhances the degree of oxidation of phenolic compounds resulting in colour changes (Xiang et al. 2018). The oxidation rate is partly suppressed by the presence of antioxidants (mainly flavonoids and ascorbic acid), however when the concentration of free radicals overpowers the concentration of the antioxidants, golden brown color of Kalparasa becomes milky white (Aparajhitha and Mahendran 2019).

Fig. 1.

Fig. 1

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Effect of fermentation condition and time on the changes in the color of Kalparasa

Organoleptic studies revealed that the taste of Kalparasa was initially sweet and later (after 3 h of atmospheric storage) it turns sour (data are not shown). Ramalakshmi et al. (2018) reported that volatile components of fermented coconut sap consist of carbonyl compounds (33.28%), aliphatic alcohol (38.99%), the carboxylic acid (9%), and ester (15.28%), whereas fresh coconut inflorescence sap contains 0.26% carbonyl compounds, 25.07% aliphatic alcohol, 5.19% carboxylic acid, and 1.3% esters. Octanoic acid, 1-butanol, 2,2-diethoxy propanoic acid and 3-methyl propane have strong aroma descriptors that include “harsh” and “rancid” (Siebert et al. 2005; Ramalakshmi et al. 2018).

Changes in vitamin C during fermentation

The major contributing factor of Kalparasa antioxidant capacity is the relative vitamin C content besides its polyphenol content. Hence, it is imperative to investigate the effect of fermentation on the vitamin C content. Initially, Kalparasa (0 h) contained high vitamin C content of 14.94 mg/100 g (Table 1). Fermentation results in a significant negative effect on the vitamin C content of Kalparasa. After 6 h of fermentation, a 25% decrease in vitamin C content was observed in Kalparasa stored under atmospheric storage condition whereas a decrease of only 4.88% in vitamin C content was observed in Kalparasa stored under refrigerated condition. Similarly, Kaprasob et al. (2017) observed the decrease in vitamin C content during fermentation of cashew apple juice due to the action of lactic acid bacteria. It was due to the degradation in the activity of ascorbic acid oxidase and depletion of residual oxygen and when there is conversion to low anaerobic or micro-aerobic phase (Adetuyi and Ibrahim 2014).

Changes in total sugar and reducing sugar during fermentation

Fresh Kalparasa has 13.17% total sugar and 0.27% reducing sugar (Table 1). Results revealed that the concentration of total sugar decreased gradually whereas reducing sugar increased as the fermentation time increases in atmospheric storage condition (Table 1). Refrigerated storage condition of 5 °C maintained the total sugar and reducing sugar content without any significant differences. It could be due to the fact that most of the acetic acid-producing bacteria do not grow below 8 °C (Ghosh et al. 2012). Around 25.96% decrease in total sugar content was observed during the course (0 to 6th hour) of fermentation in atmospheric storage conditions. During the first 3 h of storage, the degree of conversion rate from total sugars to monomers was comparatively less. During the last 3 h of storage, the conversion of total sugar to reducing sugar remained faster. i.e., total sugar content decreased by 16.87% (11.73% at the 3rd hour and 9.75% at 6th hour) and reducing sugar content increased to 72% (0.54% at the 3rd hour and 0.93% at 6th hour). Changes in sugar content could be due to the conversion of sucrose to monomers (glucose and fructose) by the action of microorganisms and yeasts (Kalaiyarasi et al. 2013). Hebbar et al. (2015) reported that Kalparasa has 12–15% of sucrose and a minor amount of fructose and glucose. During the natural fermentation process, there was an inversion from sucrose to glucose and fructose. Similarly, Ramalakshmi et al. (2018) reported that half of the total sugars of fermented palm sap are converted to monomers during the first 24 h and the ethanol content reaches 5.0–5.28% after 48 h of fermentation. However, the ethanol concentration was 4.1% at 13 h of fermentation (Shetty et al. 2017) and 90 g.kg−1 on the 7th day of fermentation (Atputharajah et al. 1986). The fermentation temperature and time have a significant (p < 0.001) effect on the reducing sugar content (Table 2).

It is important to identify the microorganism involved in the fermentation process to understand the biochemical nature of sugar conversion in Kalparasa. Various types of microorganisms (aerobic mesophils) and yeasts were supported by Kalparasa due to its sugar content (Ogbulie et al. 2007). Within 6 h, population of bacteria and yeasts were observed to grow in Kalparsa samples kept under ambient and refrigerated conditions. The population count at 0 h sampling was almost similar in both conditions. However, with hourly intervals, an increase in population in both the conditions was recorded. But between the conditions, the ambient samples had close to log 2 values higher cfu than the samples under refrigerated conditions (Fig. 2, 3). Colonies in the nutrient agar plates initially selected the bacterial growth until 2 hourly sampling and then more yeast colonies started growing whereas, Sabouraud Dextrose Agar (SDA) plates selected yeasts alone. No mold growth was observed. By the last sampling (6th hour), growth of yeasts colonies was fully covering the SDA plate and they were too many to count. Hence, the bacterial populations are dominating the initial fermentation period followed by yeasts growth. Atputharajah et al. (1986) found a total of 39 isolates of bacteria and 166 isolates of yeasts from the coconut sap during its natural fermentation. Yeasts and lactic acid bacteria are playing a predominant role in the fermentation of saps. Ramalakshmi et al. (2018) highlighted that when fermentation duration increases total bacterial count decreases whereas the count of mold and yeast increases. Shetty et al. (2017) reported that the lactic acid bacteria were predominant up to 7th hour of fermentation (8–12 × 107 cfu/ml) and the count decreased to 2.5 × 107 cfu/ml at the end of 13th hour. However, the yeasts count increased from 3.4–4.4 × 107 (at 7th hour) to 8.75 × 107 at the end of 11th hour fermentation. Yeasts are responsible for the hydrolysis of reducing sugars to alcohol. Scientific reports revealed that the production of alcohol and acid in the medium phase of fermentation control bacterial growth. At the same time, mold and yeast are best suited to survive on alcoholic and acidic conditions than bacteria (Ramalakshmi et al. 2018).

Fig. 2.

Fig. 2

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Time course analysis of bacterial population in Kalparasa stored under ambient (Non-Ref) and refrigerated (Ref) conditions

Fig. 3.

Fig. 3

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Time course analysis of yeast population in Kalparasa stored under ambient (Non-Ref) and refrigerated (Ref) conditions

Kinetics of physico-chemical parameters

The pH, vitamin C and total sugar in Kalparasa were reduced with the increase in fermentation time (Table 1). Corroborating the earlier reports (Shetty et al. 2017; Ramalakshmi et al. 2018), a higher fermentation time caused a lower pH, vitamin C and total sugar content in Kalparasa. To further investigate the degradation mechanism of these three physico-chemical properties during the fermentation process, the zero, first, and second-order kinetic models were applied to predict the rate of degradations.

The reaction kinetics models could predict better when the standard error (SE) and root mean square error (RMSE) values are less and coefficient of determination (R2) value was closer to one (Pandiselvam et al. 2017b, 2018), while the kinetic model is acceptable, if the R2 value > 0.90 (Li et al. 2020). The R2 values for pH, turbidity, acidity, and reducing sugar were ≥ 0.90 in all the three models (Table 3), suggesting that the kinetic models adequately describe the acidity production during fermentation of Kalparasa. But the SE and RMSE value for turbidity were less in second-order equation (in both the fermentation conditions) compared to zero and first-order equations (Table 3). Whereas, the SE and RMSE value for reducing sugar is best fit in first-order equation (in both the fermentation condition). Previous studies indicated that the degradation of phytochemical content (total phenolic and flavonoids) and antioxidant capacity during the fermentation of cabbage juice followed the first-order reaction kinetics (Jaiswal and Abu-Ghannam 2013). Similarly, Ou et al. (2009) found that the thermal degradation of monacolin in Monascus-fermented solution followed first-order or second-order kinetics. The difference in the kinetic models may be due to the fermentation conditions (temperature, initial microbial load, pH of the substrate, and biochemical constituents). The R2 values for TSS, vitamin C and total sugar were in the range of 0.62–0.83 (Table 3). The very low R2 value of 0.00052–0.00058 and 0.36–0.37 was observed for the attributes TSS and vitamin C of the Kalparasa stored under refrigerated conditions, respectively (Table 3), reflecting the poor model performance. Thus, these three kinetics models were not suitable to describe and predict the changes in TSS and Vitamin C value during low-temperature fermentation.

Table 3.

The kinetics parameters obtained for the physico-chemical properties of Kalparasa during fermentation

Atmospheric storage Refrigerated storage
Variables K (hour−1) R2 SE RMSE t1/2 (hour) K (hour−1) R2 SE RMSE t1/2 (hour)
Zero-order
 pH 0.571 0.92 0.075 0.337 5.86 0.014 0.35 0.009 0.039 239.88
 TSS 0.088 0.83 0.018 0.08 – 0.002 0.00052 0.047 0.209 –
 Turbidity 120.342 0.86 21.55 96.354 – 1.814 0.96 0.176 0.786 –
 Acidity 0.047 0.83 0.009 0.042 – 0.004 0.74 0.001 0.005 –
 Vitamin C 0.567 0.62 0.197 0.882 13.18 0.32 0.36 0.189 0.845 23.2
 Total sugar 0.452 0.68 0.139 0.619 13.86 0.395 0.64 0.133 0.593 16.375
 Reducing sugar 0.102 0.93 0.013 0.057 – 0.01 0.69 0.003 0.014 –
First-order
 pH 0.112 0.91 0.015 0.07 6.19 0.002 0.35 0.001 0.006 346.5
 TSS 0.006 0.83 0.001 0.005 – 0.0001 0.00055 0.003 0.014 –
 Turbidity 0.64 0.92 0.083 0.374 – 0.057 0.97 0.005 0.021 –
 Acidity 0.514 0.90 0.075 0.338 – 0.138 0.79 0.032 0.144 –
 Vitamin C 0.042 0.62 0.014 0.066 16.5 0.025 0.37 0.014 0.065 27.72
 Total sugar 0.04 0.68 0.012 0.054 17.33 0.035 0.63 0.011 0.053 19.8
 Reducing sugar 0.17 0.93 0.020 0.090 – 0.029 0.67 0.009 0.04 –
Second-order
 pH 0.022 0.9 0.0034 0.015 6.79 0.0003 0.35 0.0002 0.001 744.42
 TSS 0.0004 0.83 0.0001 0 – 0 0.00058 0.0002 0.001 -
 Turbidity 0.007 0.91 0.001 0.004 – 0.0018 0.97 0.0001 0.001 -
 Acidity 9.297 0.93 1.141 5.101 – 4.9362 0.81 1.061 4.743 -
 Vitamin C 0.003 0.62 0.0011 0.005 22.3 0.0019 0.37 0.0011 0.005 61.23
 Total sugar 0.004 0.68 0.0011 0.005 19.95 0.003 0.62 0.0011 0.005 70.27
 Reducing sugar 0.3 0.93 0.0373 0.167 – 0.0827 0.66 0.0265 0.119 -
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K, rate constant; R2, coefficient of determination; SE, standard error; RMSE. root mean square error; t1/2, half life

The kinetic parameters such as reaction rate constant (k) and half-life (t1/2) of physico-chemical attributes during the process of fermentation are summarized in Table 3. The k-values ranged from 0.0019 to 0.567 (h−1) and 0.003 to 0.452 (h−1) for vitamin C and total sugar degradations, respectively. Besides, the k-value was inversely proportional to the t1/2 value. It was found that vitamin C and total sugar are more stable under refrigerated and/or lower temperature fermentation, as indicated by lower k and higher t1/2 values. As the second-order model was applied, the t1/2 values of vitamin C degradation decreased significantly from 61.23 to 22.3 h as the temperature condition was increased from 5 °C (refrigerated) to 31 °C (ambient). A similar tendency with lower t1/2 values of vitamin C degradation was observed following the application of first-order model, where t1/2 values decreased from 27.72 to 16.5 h with an increase in temperature from 5 to 31 °C. Total sugar degradation also followed similar kinetics. The k value of total sugar [14.43% (in zero-order) 14.28% (in first-order) and 33.33% (in second-order)] was high in atmospheric fermented Kalparasa, than refrigerated fermentation. The t1/2 values for the total sugar degradation in atmospheric fermentation ranged from 13.86 h to 19.95 h, which was less than the values observed in low-temperature fermentation (16.375–70.27 h), indicating that the degradation of total sugar is dependent on the fermentation temperature. The optimum temperature (30–35 °C) would enhance the microbial growth in the Kalparasa causing rapid degradation of total sugar.

Conclusion

Kalparasa is a highly nutritive drink, but its rapid fermentation nature encounters difficulties in storage and marketing. The present study was planned to understand the shelf life of Kalparasa under different storage conditions. Also, the optimization of fermentation conditions is important to produce various products including vinegar and ethanol with better characteristics and required end-use. The pH and TSS of the sap declined to 3.74 and 14.87 °Brix, respectively, after 6 h of atmospheric fermentation. The reducing sugar content recorded a maximum of 0.93% at the 6th hour of atmospheric storage when compared with 0.27% at the 0th hour. Kinetic models were applied to simulate the acetic acid production and vitamin C and total sugar degradation. First-order and/or second-order reaction models are found to be useful for the prediction of shelf life of Kalparasa and also to simulate the rate of production of end products including vinegar and ethanol.

Availability of data and material

All the data were presented in Tables and Graph Format.

Acknowledgements

Authors are grateful to Indian Council of Agricultural Research (ICAR) for the financial support for the research. The support provided by The Director, ICAR-CPCRI is highly appreciated.

Authors' contributions

RP: Conceptualization; Methodology; Original draft writing. MRM: Writing—review & editing; Supervision. SMB, SVR, SB and MG: Methodology; Writing—original draft. KBH and ACM: Project administration; Resources. AK, RK and SS: Reaction kinetics & Modelling.

Funding

This work was funded by Indian Council of Agricultural Research (ICAR). Grant Number: 1000767018.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

Ethical approval was not required for this research.

Footnotes

Publisher's Note

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Contributor Information

R. Pandiselvam, Email: anbupandi1989@yahoo.co.in

M. R. Manikantan, Email: manicpcri@gmail.com

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