Thahira Banu A,Sri Ramani P,Aswini Murugan

School of Sciences,Department of Home Science,The Gandhigram Rural Institute-(Deemed to be University),Gandhigram,624302,Dindigul,Tamil Nadu,India

ABSTRACT Seaweeds are the natural marine algae which have several properties to act as natural preservatives to extend the shelf life of perishable foods without affecting their quality and causing side effects.Locally available and cheap fruits such as tomato are perishable very easily due to its high water content.Seaweed gel coating is a good choice,easily available and cheap natural preservative abundantly available in the Indian coasts.The present study was undertaken to extend the shelf life of the tomatoes using seaweed gel coating at different concentrations.Two seaweeds namely Kappaphycus alvarezii(red algae)and Sargassum tenerrimum(brown algae)was selected due to their high gelling property.Selected seaweeds were collected in dry form from Aqua Agri Processing Limited,Mana Madurai (SIPCOT) Tamil Nadu,India.The seaweed extracts were tested for phytonutrients,antibacterial and anti-fungal activity using standard procedures.The shelf life of seaweed coated tomatoes using different concentrations of the seaweed extract(gel)was tested.From the results,it is understood that the phytonutrients namely flavonoids,steroids,saponins and tannins are present in both the selected seaweeds.Antibacterial and antifungal activity of Kappaphycus alvarezii showed higher inhibition zone than S.tenerrimum against common pathogens.Quality characteristics and shelf life evaluation was based on water loss,texture,ascorbic acid content,juice content,total soluble solids (TSS),total acidity and TSS/total acidity ratio,respectively.The results pointed out that K.alvarezii was the most effective in maintaining the quality characteristics of tomato fruits and it also supports the fruit quality during storage period than S.tenerrimum.Thus,from the results of the study it is concluded that the better quality characteristics and shelf life of tomatoes can be increased by coating seaweed gel and it can be used as a bio preservative in fruit and vegetable preservation.

Keywords:Seaweeds Preservatives Antibacterial Antifungal Phytonutrients Inhibition

1.Introduction

Food preservation is one of the oldest technologies used by human beings to avoid spoilage.Preservation can be carried out by using natural and chemical preservatives.Preservatives are the substance required to support quality,extend the shelf life and make sure the safety of fresh and processed food products[1].Natural substance act as a preservative in food to prevent spoilage,the common natural preservatives are salt,sugar,vinegar,alcohol,etc.Among these,salt and sugar are most commonly used preservatives and is found to inhibit the growth of bacteria in food.Artificial or chemical preservatives commonly used are sodium benzoate,benzoic acid,sodium nitrite,sulphur dioxide,and sodium and potassium sorbate which can cause high blood pressure,increase cholesterol level and also affects the kidney and liver function[2].

Recent years’extracts of plant and marine sources are used as an alternative for artificial preservatives,such as benzoic acid,nitrite and sulphur dioxide.Natural preservatives obtained from marine sources.Seaweeds are the natural marine algae that possess several properties as a natural preservative to extend the shelf life of the perishable foods without causing any side effects.Seaweed or macroalgae are aquatic plants belonging to the plant kingdom thallophyta[3].Seaweeds are primitive non-flowering plants without true root stem and leaves.These organisms constitute a total of 25-30000 species with great diversity[4].Seaweeds are classified into three broad groups based on pigmentation namely brown algae(Phaeophyta),red algae (Rhodophyta) and green algae (Chlorophyta)[5].

Seaweeds contain 80-90 percentage water and their dry weight has 50%carbohydrate,1%-3%lipids,and 7%-8%minerals.The protein content is highly variable (10%-47%) with high proportions of essential amino acids [6].Seaweeds are known for their richness in polysaccharides,minerals,omega 3 and 6 polyunsaturated fatty acids(PUFA),vitamins and some bioactive substances,such as polyphenols which it contain antihyperlipidemic,antihypertensive and antibacterial properties[7].

The huge amount of fruits and vegetables is degraded during the post-harvesting process.Locally available and cheap fruit such as tomato is perishable very easily than other fruits due to increased water content.Usage of chemical preservatives to improve the keeping quality of fruits and vegetables was expensive and leads to several health hazards to humans.Tomato fruit is classified as a berry.Tomatoes (Lycopersion esculenntummill) are a popular and versatile food that comes in over one thousand varieties.Tomatoes are not only a good source of vitamins A and C,but also a good source of vitamins and minerals.Tomatoes contain higher levels of phosphorus,potassium,folate,antioxidants,beta-carotene,and lycopene.Tomatoes are all time available fruits easily perishable.Spoilage of tomatoes causes adverse quality changes that include the change in taste,smell,appearance or texture of the tomatoes[8].So,Natural preservation with a coating of seaweed gel was found to be a good choice.Availability,cost-effective,lesser or no side effects and ability to increase shelf life of the tomatoes,with this background the present study was undertaken to analyze the phytonutrient composition,antimicrobial activity of the selected seaweed and to understand the shelf life of the tomatoes using seaweed gel coating.

2.Methodology

2.1.Selection and collection of seaweeds

Two seaweeds namelyKappaphycus alvarezii(red algae)andSargassum tenerrimum(brown algae) have been selected due to its gelling property,antibacterial activity is due to help in the preservation of fruits and vegetables.Selected seaweeds were collected in dry form from Aqua Agri processing limited,Mana Madurai(SIPCOT),Tamil Nadu,India.

2.2.Preparation of seaweed extracts

Two seaweeds namelyK.alvareziiandS.tenerrimumwere subjected to boiling for extracting the gel.Five grams of the seaweeds were taken and immersed in 500 mL of water and boiled for 15 min at 100°C.After cooling,it was filtered and allowed to set at room temperature((25±2)°C).Further,the gel-like liquid obtained on cooling was used to analyze the phytonutrient composition,antibacterial activity and taken in different concentration for coating on tomatoes for extending its shelf life.

2.3.Determination of phytonutrient composition

Aqueous extracts ofK.alvareziiandS.tenerrimumwere subjected to a preliminary screening of different phytonutrients such as alkaloids,terpenoids,steroids,tannins,saponins,flavonoids,phlorotannins,glycosides and cardiac glycosides using standard procedures described by literature [9].Further,the extract was subjected to quantitative phytonutrients such as Total Flavonoids Content(TFC)and Total Phenolic Content(TPC)using standard procedures described by literature[10].

2.4.Determination of total flavonoids content

Total flavonoids content(TFC)of the seaweed extracts was measured by using the colorimetric method described by literature[10].The extract(0.5 mL)was mixed with 2.25 mL of distilled water in a test tube followed by the addition of 0.15 mL of 5%NaNO2solution.After 6 min,0.3 mL of 10%Alcl3.6H2O solution was added and allowed to stand for another 5 min and 1.0 mL of 1 mol/L NaOH was added.The mixture was mixed well using a vortex.The absorbance of the final mixture was measured immediately at 510 nm.The result was expressed in terms of mg rutin equivalents in 1 g of the dried sample(mg RE/g).

2.5.Determination of total phenolic content

Total phenolic content(TPC)of the selected seaweed was determined using the folin-ciocalteu reagent was measured by using the colorimetric method followed by literature [10].The extract (0.3 mL)was made up to 1 mL with distilled water and mixed with 2.25 mL of folin-ciocalteu reagent diluted (1:10) in distilled water and allowed to stand at room temperature for 5 min.Sodium carbonate solution (2.25 mL) was added to the mixture.After 90 min at 28°C,the absorbance was measured at 725 nm.The total phenolic content was calculated and expressed as gallic acid equivalent per gram of dry weight (mg GAE/g DW) based on gallic acid (100 μg/mL)standard curve.

2.6.Determination of antimicrobial activity of the seaweed extracts

Extract of the two selected seaweed namelyK.alvareziiandS.tenerrimumwas subjected to antimicrobial activity using a well diffusion method.The common microorganisms that bring out spoilage in tomatoes were identified and the antibacterial activity of the extracts towardsBacillus subtilis,Staphylococcus aureus,Lactobacillus acidophilus,Escherichia coli,Pseudomonas aeruginosaandProteus mirabilisand antifungal activity of the extract towardsAspergillus flavus,Aspergillus niger,Aspergillus furigate,Candida albicans,andCandida tropicaliswere studied and the zone of inhibition was determined.

The bioassay was carried out using the agar disc diffusion method with a paper disc of 6 mm diameter,prepared from Whatmann No.1 filter paper.The bacteria inoculated were grown in nutrient broth overnight and a fixed volume was inoculated into 10 mL aliquots of nutrient agar,mixed and then poured over a nutrient agar sterile Petri dishes.This formed the bacterial lawn.Initially,both paper discs were used for testing the seaweed extracts.The paper disc of 6 mm soaked in 6 mL of seaweed extracts and places onto the bacterial lawn after it had solidified,the standard antibiotic disc was incubated at 37°C overnight.The zones of inhibition were measured after 24 h of incubation[11].

2.7.Determination of antifungal assay

Young fungal cultures were incubated for 2-3 days at room temperature and seeded on Sbouraud Dextrose Agar (SDA) plates for bioassay by agar disc diffusion method.Whatmann No.1 filter paper disc of 6 mm containing the seaweed extract was placed on the surface of the plates.After 72 h at 30°C,the plates were observed for the presence of inhibition zones[11].

2.8.Evaluation of the seaweed coating on shelf-life of tomatoes

Selected fruits were washed,air-dried and used for seaweed coating in different concentrations.For treatment 84 healthy uniform-sized tomatoes were selected and grouped into three with 36 tomatoes in two experiment groups coded as an experiment I and II treated withK.alvarezii and S.tenerrimumrespectively.The control group had 12 tomatoes and not treated with any coating.The selected seaweed extract(gel)was prepared in three different concentrations of 1%,2% and 3% and were used for coating.The following parameters were evaluated.

2.8.1.Weight loss

Weightloss canbecalculated fromthe difference betweeninitial and final weight and expressed as zero percent of the initial fresh weight[12].

2.8.2.Texture

Textural changes in firmness and size of tomato between the initial and final stage per each sample for each treatment was studied.

2.8.3.Total soluble solids(°Brix)

Total soluble solids (TSS) was determined using hand refractometer instrone,Brix reading range between 0%-40%[12].

2.8.4.Total acidity

Total acidity was determined in fruit juice as citric acid content as described in[13].

2.8.5.Total soluble solids/total acidity ratio

Total soluble solids/total acidity ratio were recorded by dividing TSS value by total acidity value[14].

2.8.6.Ascorbic scid content

Ascorbic acid content was determined using 2,6-dicholorophenol indophenols dye by titration methods as described by[13].

2.8.7.Juice percentage

Tomato juice was extracted using a blender and weighed and calculated as the percent of fruit weight for each treatment as given by literature [14].Juice content is calculated using the following method

2.9.Statistical analysis

All the investigations were carried out in triplicate and mean±standard deviation were calculated.

3.Results and discussion

3.1.Phytonutrient composition of the selected seaweed extracts

Non-nutritive substances that are naturally present in plant are known as phytonutrients.They provide many beneficial health properties as they have the potential to modulate human metabolism that favors prevention of disease[15].

Table1 Total flavonoid and total phenolic content of the seaweed extracts.

3.2.Qualitative and quantitative phytonutrient composition of the selected seaweed extracts

From the results of qualitative phytonutrient composition,it was clear that the tannins,flavonoids,alkaloids,steroids,and saponins were present in both extracts whereas phlorotannins and terpenoids were present inS.tenerrimum,anthroquinones,glycosides and cardiac glycosides were absent in both extracts.Anthroquinones,cardiac glycosides,alkaloids,and terpenoids failed to show their presence in any of the tested extracts ofS.duplicatum[16].The presence of phytochemical in ethanol,methanol and acetone extracts ofK.alvarezii[17].The results revealed the presence of various phytochemical constituents namely alkaloids,phenolic compounds,flavonoids,terpenoids and tannin in all the extracts used.The methanolic extract showed a maximum number of active phytochemicals compared to ethanolic and acetone extracts.

Quantitative phytonutrient composition in Table1 reveals that the total flavonoid and total phenolic content inK.alvareziiwas found to be (0.2143 ± 0.03) mg/g and (0.4549 ± 0.001) mg/g andS.tenerrimumwas found to be(0.4549±0.02)mg/g and(0.3110±0.001)mg/g.K.alvareziishowed a slightly less amount of flavonoid content compared toS.tenerrimum.The brown algaeDichotomashowed higher phenolic content((16.375±0.44)mg GAE/g)than the selected red seaweeds[18].

3.3.Antibacterial activity of the seaweed extracts

Antibacterial activity was detected using aqueous extract ofK.alvareziiandS.tenerrimumand the results are presented in Table2.It is evident from the table that seaweed extracts have antibacterial activity.Among two selected seaweed extracts,K.alvareziihad the highest inhibition zone at 100 μg/mL concentration againstBacillus subtilis,Staphylococcus aureus,Lactobacillus acidophilius,Escherichia coli,Pseudomonas aeruginosa,andProteus mirabilis.At lower concentrations(80 μg/mL)K.alvareziihad higher inhibition againstL.acidophilius,E.coli,andP.mirabilis.S.tenerrimumextract also had a considerable antibacterial activity againstB.subtilis,S.aureus,E.coli,P.aeruginosa,andP.mirabilis.The antibacterial activity was higher for the standard with 16.97-21.03 mm at 100 μg concentration.

The antibacterial activity ofK.alvareziiagainstSalmonella typhi,Bacillus spp,S.aureus,E.coli,Proteus,Pseudomonas,andStreptococcus pyogenesandCandida albicanshad the highest inhibition zone at 100 μg/mL concentration[19].

The antimicrobial activity chloroform,ethanol,methanol and water extracts ofS.tenerrimumextract at different concentration of 100,300 and 500 mg/mL.Among the four extracts taken ethanol extract had the highest antibacterial activity againstS.aureus(15 mm) in the concentration of 100 mg/mL.While at 500 mg/mL chloroform extract had promising activity against pathogens likeS.aureus(17 mm),S.anginosus(18 mm) andM.racemosus(13 mm) inhibition for ethanol extract.The results from the present study is understood that theK.alvarezihad the maximum inhibition zone andS.tenerrimumhad moderate inhibition zone at 100 μg/mL concentration.The antibacterial activity ofS.tenerrimumis closely related to the result obtained by literature [20].It is evi-dent from the study that bothK.alvareziiandS.tenerrimumhave promising antibacterial activity.

Table2 Antibacterial activity of the seaweed extracts.

Table3 Antifungal activity of the seaweed extracts.

3.4.Antifungal activity of the seaweed extracts

The results of antifungal activity of the seaweed extracts are presented in Table3.Antifungal activity of the selected seaweed extracts was assessed and found thatK.alvareziihad higher inhibition against the selected organisms thanS.tenerrimum.Inhibition zone was high at 40 μg/mL concentration ofK.alvareziiextract againstApergillus flavus(12 mm),Aspergillus niger(5 mm),Aspergillus furigate(11 mm),Candida albicans(8 mm)andCandida tropicalis(14 mm).S.tenerrimumhad lowest inhibition zone against the organisms.However,the standard had higher antifungal activity than seaweed extract,at the range of 12 mm-16 mm.

Antifungal activity ofS.tenerrimumof chloroform,ethanol,methanol and water extracts at 100,300 and 500 mg/mL concentrations.At 100 mg/mL,the fungal strains ofA.niger(10 mm),R.stolonifer(10 mm)andM.racemosus(10 mm)showed mild activity in four extracts.At 300 mg/mL,the fungal strain of water extracts ofA.niger(14 mm),methanol extract ofC.albicans(13 mm) and ethanol extract ofA.niger(13 mm) showed moderate activity.At 500 mg/mL,the fungal strainA.niger(14 mm),S.cerevisiae(13 mm)showed mild activity.Methanol extract observed the promising activity against pathogens likeE.coli(15 mm),P.vulgaris(15 mm),C.albicans(14 mm)andR.Solani(10 mm)showed low activity.Water extract observed promising activity against pathogens likeE.coli(14 mm),P.vulgaris(14 mm),E.aerogenes(14 mm)andA.niger(15 mm),R.stolonifer(14 mm).

The result of the present study indicates thatK.alvareziihad better inhibition zone at 60 μg/mL concentration thanS.tenerrimum.The results of the present study are in line with the findings reported by literature[20].

Table4 Weight loss of the uncoated and coated tomatoes during storage.

3.5.Effect of seaweed coating on quality characteristics and shelf life of tomatoes

Effect of seaweed coating on quality characteristics and shelf life of tomatoes based on weight loss,texture,ascorbic acid content,juice content,total soluble solids(TSS),total acidity,TSS/total acidity ratio of the control and seaweed coated tomatoes during storage are presented below.

3.6.Weight loss of the uncoated and coated tomatoes during storage

Weight loss of both control and seaweed coated tomatoes were recorded on storing for 28 days.The results of weight loss during the storage period were presented in the following Table4 and Figure 1.Weight loss was recorded in tomato fruits coated and uncoated with different concentrations ofK.alvareziiandS.tenerrimumextract coating.Tomato coated with 3%K.alvareziiextract contained the lowest level of weight loss,followed by 3%S.tenerrimumextract had a low level of weight loss compared to other concentrations coating and the control.Uncoated tomatoes showed the highest weight loss compared to other treatments.Coating 3%K.alvareziiextract was more effective in maintaining the weight of the tomatoes.The edible coatings provide an effective barrier to oxygen,carbon dioxide,and water vapor transmission thus helping to alleviate the problem of moisture loss [21].The findings of the present study were found to be in line with the study reported by literature [22]that minimum weight loss with chitosan and carrageenan coatings in strawberries(Fig.1).

3.7.The texture of the uncoated and coated tomatoes during storage

The texture of both control and seaweed coated tomatoes were recorded on storing for 28 days.The changes in texture during the storage period are presented in Table5 and Plate 1.It is evident from the results that the texture of the seaweed coated tomatoes was firm and fresh than the control.It is also noticed that as the concentration of seaweed increased the firmness of the fruit increased.Texture loss is the most noticeable change occurring in tomato during prolonged storage and it is related to metabolic changes and water content.Three percentK.alvareziiextract coating was found to maintain the firmness of the fruit and increased the shelf life to 28 days.The tomatoes in control group were found to be firm only up to 14 days of storage(Plate 1).

Fig.1.Weight loss of the uncoated and coated tomatoes during storage.

Table5 Texture of uncoated and coated tomatoes during storage.

Table6 Ascorbic acid content of the uncoated and coated tomatoes during storage.

Table7 Juice content of the uncoated and coated tomatoes during storage.

3.8.Ascorbic acid content of the uncoated and coated tomatoes during storage

The changes in ascorbic acid content during the storage period are presented in Table6 and Figure 2.From the results,it was observed that during the storage period there was a drastic decrease in the ascorbic acid content of tomatoes in the control group.It was found that the application of 3%K.alvareziiextract coating retention of ascorbic acid content was considerably high during the storage period.Only a loss of 3%ascorbic acid in coated tomatoes recorded while in control group 15% loss was noticed.Similarly,3%ofS.tenerrimumextract coating also had the highest retention of ascorbic acid during the storage period.Thus it is evident that seaweed coating helps in the retention of ascorbic acid content in tomatoes(Fig.2).

3.9.Juice content of the uncoated and coated tomatoes during storage

Juice percentage of the control and seaweed coated tomatoes during the storage period are tabulated and presented in Table7 and Figure 3.The data shows the effect of seaweed coating treatments such asK.alvareziiat 1%,2%and 3%concentrate,S.tenerrimumat 1%,2% and 3% and untreated tomatoes on changes in juice percentage during the storage period (28thday).Compared with untreated fruit,3%K.alvareziiextract coated tomatoes had the highest level of juice content than 1% and 2% ofK.alvareziirespectively.Whereas 1%,2% and 3%S.tenerrimumextract coated tomatoes had the lowest volume of juice content after 28 days of storage was observed.Thus this confirms thatK.alvareziiextract coatings act as a barrier to moisture loss and therefore retarding dehydration of tomatoes(Fig.3).

Plate 1.Texture of uncoated and coated tomatoes during storage.

Table8 Total soluble solids(TSS° Brix)of the uncoated and coated tomatoes during storage.

3.10.Total soluble solids(TSS° Brix)of the uncoated and coated tomatoes during storage

It is clear from Table8 that 3%K.alvareziiextract coated tomato had the highest soluble solids content during the 28 days of storage period compared to other treatments.One percentS.tenerrimumhad the least soluble solids respectively.The total soluble solids of the tomatoes in the control group decreased drastically indicating the reduced shelf life of the fruit.

The soluble solid depends on the maturity stage and it increased generally during the ripening process due to the polysaccharide hydrolysis to maintain the respiration rate [23].The higher level of total soluble solid in the tomato coated or treated with 3%K.alvareziiextract may be due to protective O2barrier reduction of oxygen supply on the fruit surface which inhibited respiration.The decrease of total soluble solids caused by a decline in the number of carbohydrates and pectins,partial hydrolysis of protein and decomposition of glycosides into subunits during respiration.

3.11.Total acidity of the uncoated and coated tomatoes during storage

The changes to the total acidity percentage of the control and seaweed coated tomatoes during storage were presented in Table9.At the end of the storage period,untreated tomato contained the lowest total acidity value and 1%K.alvareziiextract coating had the lowest total acidity value on 28 days of storage period.Three percent ofK.alvareziiextract coating had a high total acidity value compared to other treatments.The acidity percentage of tomato fruits decreased with increase in storage period.

Fig.2.Ascorbic acid content of the uncoated and coated tomatoes during storage.

Fig.3.Juice content of the uncoated and coated tomatoes during storage.

Table9 Total acidity of the uncoated and coated tomatoes during storage.

Organic acid such as citric acid are primary substrates for respiration and reduction in acidity is expected in highly respiring fruits and coating or wrapping treatments reduce respiration rates and may,therefore delay the utilization of organic acids during storage[12].Thus,3%K.alvareziitreated tomatoes the acidity reduced only by 1%-2%.Whereas the tomatoes treated withS.tenerrimumanduntreated the reduction in acidity was high indicating the organic acid present in them are available as substrate for respiration.

Table10 TSS/total acidity ratio of the uncoated and coated tomatoes during storage.

3.12.TSS/total acidity ratio of the uncoated and coated tomatoes during storage

The effect of different concentrate (1%,2% and 3%) coating ofK.alvareziiandS.tenerrimumextract on TSS/total acidity ratio was presented in Table10.It is evident from the table that 3%K.alvareziiextract coated tomatoes had the highest TSS/total acidity ratio during the storage period of 28 days and followed by 3%S.tenerrimumcontain the highest amount of TSS/total acidity ratio.One percentS.tenerrimumextract contain least TSS/total acidity ratio compared to other treatments.In general,TSS/total acidity ratio in fruit juice significantly increased on increasing storage period[12].

4.Conclusion

From the mentioned results,it is concluded that the 3%K.alvareziiwas the most effective treatment in maintaining the quality characteristics of tomato fruits during the storage period of 30 days.Three percentK.alvareziiextract coating maintains fruit quality during storage compared withS.tenerrimumand control treatment(uncoated).Treated fruit with 3%K.alvareziiextract had the lowest percentage of weight loss and had the lowest reduction in total acidity and ascorbic acid content and juice content compared with control.Coated fruit with 3%K.alvareziiextract had a high level of TSS/total acidity ratio and good texture compared to other treatments and uncoated.Thus,from the study,it is concluded that the shelf life of tomatoes can be increased by coating with seaweed extracts.Seaweeds are promising natural preservatives that can be used in improving the shelf life of tomatoes and maintain the fruit quality.

Declaration of Competing Interest

There are no conflicting interests.