Physico Chemical Parameters of Muskmelon Juice enriched with Probiotic lactic Acid Bacteria

the spoilage of muskmelons was rapid due to early maturity of the fruits after immediate harvest before consumption. to minimise the postharvest losses, especially in horticultural crops, food processing and value addition to the produce plays lot of role and the losses can be minimised. Keeping in view, the experiment was planned to prepare muskmelon fruit juice fortified with four different species of lactic acid bacteria viz., Lactiplantibacillus plantarum subsp. plantarum MtCC 9511 , Lactobacillus acidophilus MtCC 10307 , Lactobacillus delbrueckii subsp. lactis and Lactobacillus casei MtCC 1423 . the survivability of lactobacilli and physicochemical parameters were studied during fermentation of the fruit juice. Drop in pH levels from initial pH was recorded in all muskmelon fruit juice samples incubated at two different temperatures (30°C and 37°C) more than 72 hours. But titratable acidity was increased in all muskmelon fruit juice samples incubated at two different temperatures (30°C and 37°C). the fruit juice containing Lactiplantibacillus plantarum subsp. plantarum MtCC 9511 (t4) recorded lower pH levels and maximum titratable acidity, total phenolic content and more viable cells compared to other species of lactobacilli. Sensory evaluation was conducted randomly for all the samples and no significant difference was observed.


iNtRODuCtiON
Probiotics are often called "good" or "helpful" bacteria since they help in keeping gut healthy. Probiotics have a long history of association with human beings and animals. 1 More than a century ago, Henry Tissier 2 from Pasteur Institute isolated rod shaped bifidobacteria from healthy breast fed infants and reported that these bacteria were dominated in healthy breast fed infants and they were absent from formula fed infants suffering from diarrhoea and pave the way to establish a concept that these bacteria played a role in maintaining gut health. Probiotics are defined by the World Health Organization as "live microorganisms that when administered in adequate amounts confer a health benefit on the host". 3 A probiotic food product on an average should contain 10 6 CFU/mL (colony forming units per mL) as per the guidelines set by World Health Organisation (WHO). Probiotic bacteria are conventionally added to ferment the dairy milk for production of yoghurt, dahi and other similar products making them as functional foods. Many dairy industries are commercially producing probiotic fermented products and being sold throughout the world. Few people have lactose intolerance, allergic to dairy products and presence of high saturated fatty acids in dairy foods making them unfit for consumption. 4,5 Hence, in recent times non-dairy probiotic foods are gaining importance and several raw materials like cereals, soybean, fruits and vegetables are using for production of non dairy functional foods. 6 Several probiotic strains have the ability to ferment non dairy foods like fruits and vegetables. Further, fruits and vegetables are good substrates for growth of probiotic lactic acid bacteria. 7,8,9 In the present scenario, fruit juices with probiotic bacteria are gaining more importance 10 attributed to more health benefits by consumption of fruit juice with added probiotics. 11 Lactic acid bacteria (LAB), generally and widely from the genera Lactobacillus and Bifidobacterium and constitute a maximum proportion of probiotic cultures as nutritional supplements, pharmaceuticals and functional foods 12,13 . . Cucumis melo L. belongs to Reticulatus group and commonly called as cantaloupe or muskmelon and belongs to Cucurbitaceae family. 14 This fruit is rich source of phytonutrients like sugars, ascorbic acid, carotene, folic acid and minerals and its sugar content, flavour and texture mainly decides the consumer preference. 15,16 Hubbard et al 17 hypothesised that muskmelon fruits experience a metabolic shift indicated by both physical and compositional changes, such as exocarp netting, mesocarp softening, and the commencement of sucrose accumulation, about halfway through their growth. Postharvest losses of muskmelon in some of the countries accounts for more than 30 percent or 35-40 billion income annually before its consumption after immediate harvest. 18 Muskmelons are more vulnerable to pathogenic fungi and fungal rots due to high moisture content, easily available nutrients and favourable pH and leads to unsound fruits and not fit for human consumption.
The ripening of muskmelon fruit is rapid and highly coordinated, hereditary characteristics involving a series of irreversible biochemical and physiological changes that leads to the formation of a soft and edible fruit with suited excellent features. 19 Quality of the muskmelon fruit can be judged by it's sugar composition and several key enzymes are responsible for sucrose metabolism. 20 Based on earlier studies by Hubbard et al 17 16 Because of quick ripening, the muskmelons spoils quickly within 4-5 days. Technologies to delay ripening process and proper storage facilities are yet to reach small and marginal farmers. Therefore, the present topic has been undertaken with a view to prepare value added probiotic muskmelon juice supplemented with probiotic cultures like lactic acid bacteria. To give value addition to the muskmelon fruit, maximum returns to the farmers and to curb postharvest losses during superfluity season, there is urgent need to process the excess produce into nutritious value-added products The processing of muskmelon fruits may be converted to different products like fruit leather, jams, pickles, jellies, squashes and juices etc. 18

Strains and Cultures
The

Preparation of Probiotic Muskmelon Juice
Healthy muskmelon fruits purchased from local market and washed with clean tap water thoroughly to remove extraneous material. The fruit pulp and water were mixed in 1:1 ratio and 50 g of sugar was added per kilogram of pulp for preparation of muskmelon fruit juice.
Total soluble solids (TSS) were measured by hand refractometer and it was 8%. Approximately, 150 ml of muskmelon juice is filled to conical flasks and pasteurized to remove unfavourable microorganisms. Muskmelon juice was inoculated with one ml of probiotic strains (Lactobacillus acidophilus MTCC 10307, Lactiplantibacillus plantarum subsp. plantarum MTCC 9511, Lactobacillus casei MTCC 1423, Lactobacillus delbrueckii subsp. lactis) and allowed for fermentation at 30 o C and 37 o C. To know the effect of cold storage (4 o C) on viable lactobacilli, the fruit juice samples were stored at cold conditions (refrigerated conditions i.e., 4-6 o C). All the samples in different treatments were maintained in triplicates.

Physico Chemical Analysis during the Fermentation of Muskmelon Juice
pH values for muskmelon juice samples were recorded by a digital pH meter. The titratable acidity of the fermented probiotic muskmelon fruit juice samples were determined titrating against 0.1M of sodium hydroxide (NaOH) in the presence of phenolphthalein indicator. To the 5ml of sample, add 6-7 drops of indicator and titrated against 0.1M NaOH (50ml) in burette till colour change noticed.

Viable Cell Count
Viable cell counts of lactobacilli for all juice samples were isolated on de Mans Rogosa agar (MRS agar) medium by the standard plate count (SPC) method. All the MRS plates were kept for incubation at 30°C for 48 hours to check the lactobacilli colonies. 21

Quantitative Analysis of Carbohydrates
Carbohydrates like glucose, fructose and sucrose HPLC (Shimadzu model LC6A) according to Casterline et al. 22 Ten μl (10 μl) of sample extract was injected to HPLC column to detect the concentration of sugars in the samples. Carbohydrates like glucose, fructose and sucrose are separated by isocratic method with mobile phase consists of acetonitrile: water (80:20 (v/v)) at a flow rate of 1 ml/min. The flow rate was kept at 1 ml/min. Standard sugars (sucrose, fructose and glucose) were used for calibration of HPLC and standards were purchased from Sigma chemicals, USA.

total Phenolic Concentration (tPC)
Total phenols in the probiotic fruit juice samples was determined using the Folin-Ciocalteu method, as described by Anand et al. 23 Diluted juice samples (0.5 ml) were pipetted into test tubes and 5 ml of distilled water is added followed by 0.5 ml of Folin-Ciocalteu reagent, and the mixture was kept for reaction for 3 minutes, later, 1.0 ml of 20% sodium carbonate (Na 2 CO 3) solution was added and mixed well and allowed to stand for one hour at room temperature for colour development. Absorbance was measured at 750 nm using a spectrophotometer (MODEL UV-10, Thermo Fisher Scientific, USA) and was expressed as milligrams of gallic acid equivalent per 100 ml). Standard graph was plotted by using different ranges of standard solutions of gallic acid.

Sensory evaluation
The sensory evaluation was carried for probioticated muskmelon juices by using 9-point hedonic scale 24 to know the consumer preference sensory characteristics like taste, aroma, flavour, colour and overall acceptability. Muskmelon juice samples which were stored at refrigerated conditions for four weeks were randomly served for sensory evaluation. The average mean scores of all the sensory parameters were calculated and presented in the results.

Statistical Analysis
The treatments were kept in three replications and the experimental results were subjected to statistical analysis by one-way ANOVA. The analysis of variance and interpretation of the data was done as per the methods given by Gomez and Gomez. 25 ReSultS AND DiSCuSSiON

effect of lactic Acid Bacteria on Physical Parameters of Probiotic Muskmelon Juice
The pH levels in all fruit samples were slowly decreased during fermentation by lactic acid bacteria (Figure 1). The initial pH was recorded as 4.51 for all samples and the pH was slowly decreased in all probiotic muskmelon juice samples during fermentation by lactobacilli incubated at two different temperatures ( Figure 2).  All four probiotic cultures (Lactobacillus acidophilus MTCC 10307, Lactiplantibacillus plantarum subsp. plantarum MTCC 9511, Lactobacillus casei MTCC 1423, Lactobacillus delbrueckii subsp. lactis) were effective in reducing pH of the muskmelon juice samples. The nutrients available in muskmelon juice were utilized by all lactic acid bacteria effectively for cell metabolism and produced more organic acids after hours of incubation and this could be the reason for lowering in pH levels of muskmelon juice 4 . However, some studies reported that lactic acid bacteria became more sensitive to lower pH levels of 2.5-3.7 26 . Many fruit juice samples inoculated with lactic acid bacterial cultures especially Lactobacillus acidophilus MTCC 10307 recorded higher viable cell counts and leads to lowering of pH in juice samples with more acid production. The results are in accordance with other fruit juices like pomegranate 27 , peach 28 , mango 29 and apple juice. Acid tolerance by probiotic lactic acid bacteria in probioticated fruit or vegetable juice sample is an important trait. In general, fruit and vegetable juice fermentations carried with probiotic lactic acid bacteria have resulted in decreased levels of pH and increased levels of acidity. Lowering in titratable acidity in the probiotic muskmelon juice may be attributed to the rapid consumption of available carbohydrates by the probiotic bacteria and releasing the end products in the medium. Probiotic bacterial cultures have potential ability to grow in the fruit and vegetable juices and they have the ability to with stand the stress conditions that exist in fruit juices like acidic environment.

Sugar Consumption and total Phenolic Concentration
Fruit juice samples have total sugar content of 9-12 g/100 ml/kg. Simultaneously, simple sugars were released from the juice due to saccharification, so that these simple sugars favours bacterial growth. At  Fermentation of muskmelon fruit juice samples with lactic acid bacteria resulted significant increase in total phenolic content (TPC) and this increase may be attributed to the formation of formation of biochemical compounds like ascorbic acids, carotenoids and other phenolics during fermentation, which have been reported to have many beneficial health properties. These results have close proximity with raw or non probioticated sapota juice 28 . In general, DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging activity was significantly higher in probioticated sapota fruit juices compared to probioticated fruit juices.

Viable Cell Counts at Different time intervals
The fermented juice samples were incubated for 72 hours at two different temperatures 30 o C and 37 o C to know the survivability of lactic acid bacteria (viable cell counts). Increase in viable cell counts in juice samples which were incubated at both temperatures 30 o C and 37 o C up to 48 hours and decline in cell counts was observed after 72 hours of incubation and did not fall below 10 6 cfu/ ml ( Figure 5).
Decline in viable cell counts were noticed in the juice samples after 72 hours of fermentation  and this reduction may be due to consumption of available nutrients by the cells, reduction in pH and oxygen levels during stationary phase and occurrence of autolysis of cells in death phase 36 . Significant number of probiotic populations in finished product plays key role to confer better health benefits. The ability of probiotic lactobacilli to with stand stressful conditions like acidic environments is an important trait to maintain optimum viable counts in probiotic fruit juice to confer the desired health benefits.  38,39 . Minimum recommended probiotic population should be at least greater than 10 7 CFU/mL at the end of shelf life to confer the health benefits , [40][41][42][43] . The effect of cold storage temperatures on survivability of L. acidophilus MTCC 10307 in probiotic yogurt samples after 20 days of storage revealed that the more viability was found at 2ºC, whereas viability of B. lactis has the highest viability at 8ºC 31 . Saccaro et al 33 recorded reduction of L. acidophilus MTCC 10307 in the range of 2 log 10 cycles /mL at the end of the storage period in yogurt changes like taste, appearance and overall acceptability of Figure 6. Sensory evaluation of probioticated and non probioticated muskmelon fruit juice the product have no change and also no adverse effects were noticed. Further, L. acidophilus MTCC 10307 it is able to survive till the consumption of the product 44 .

Sensory evaluation
Probioticated muskmelon juices recorded good sensory scores and overall acceptability. significant differences was not found among the treatments regarding sensory scores of probioticated muskmelon juice samples ( Figure 6) and the influence of fermentation by lactic acid bacteria on juice texture, taste, flavours and overall acceptance was non significant. Earlier works on probiotic fruit juices like pineapple juice containing Lactobacillus reuteri, 7 apple juice containing Lactobacillus casei MTCC 1423 45 reported that these lactic acid bacteria did not affect sensory evaluation and overall acceptability. Similarly, the results are in close conformity with the results from36 who recorded that carrot flavoured milk remained in healthy condition for 4 days under cold storage. The present studies revealed that the fruit juices can be prepared with lactic acid bacterial cultures and named as probioticated fruit juices for health benefits especially to the people who have lactose intolerance and allergic to milk products.
Briefly, the results showed that the fruit juice containing Lactiplantibacillus plantarum subsp. plantarum MTCC 9511 (T4) recorded lower pH levels (2.95) and maximum titratable acidity, total phenolic content and more viable cells compared to other species of lactobacilli. In fruit juice, the contents of compounds namely glucose, fructose and sucrose were decreased as time of treatment increased.
In present experiment, the treatments with Lactiplantibacillus plantarum subsp. plantarum MTCC 9511 showed higher sensorial acceptability whereas lower sensorial acceptability was observed in treatments treated with Lactobacillus casei MTCC 1423.