A Study on Different Hurdle Factors of Nano Metal Vitamin Complex for the Prevention of Microbial Spoilage in Seafood

Hurdles that have a positive effect by inhibiting microorganisms may have a negative one on other parameters such as nutritional properties or sensory quality, depending on their intensity. In order to lower the preservative level, the hurdle technology concept has been developed, consisting in using combined hurdles to establish an additive antimicrobial effect, and even sometimes a synergetic one, thus improving the safety and the sensory quality of food. The antibacterial and anti-oxidant potential of copper oxide nano particles coupled with vitamin-E (CuONPs+VE) was investigated by applying the novel hurdle factors against seafood pathogens and by studying the cell viability using MTT assays. The hurdle method is proposed to explain the significance of combined use of different preservation factors as synergistic effects instead of using a large-intensity preservation factor. Effect of CuONPs+VE and chilling temperatures (-18◦C and +4◦C); and Effect of CuONPs+VE and brine salts at various concentrations (5%, 10%,) were evaluated. CuONPs+VE with different chilling temperatures and brine salt concentration showed signi icant results on compared to control temperatures. Thus CuONPs+VE due to their bacteriostatic activity can be ef iciently used in hurdle technology which reduces the food spoiling organisms. Thus CuONPs+VE in combined with hurdle technology can be used as alternatives for chemical preservatives in preservation techniques.


INTRODUCTION
Spoilage of seafood products is mainly due to enzymatic autolysis, oxidation and microbial growth.
Proper handling, pre-treatment and preservation techniques can improve nutritional value, texture, lavour, freshness, quality in ish and ish products (Ghaly, 2010) . Hence, low temperature storage, salting, canning, drying and chemical preservation for controlling water activity, enzymatic, oxidative and microbial spoilage are considered as common preservation methods (Abbas et al., 2009) .
Oxidative deterioration of lipids in seafood leads to the production of volatile compounds causing rancidity, changes in aroma and off-lavour production during the storage conditions. Lipid oxidation during storage is reported to be in luenced by different intrinsic factor which leads to evident colour and odour changes in ishes (Simat et al., 2017). (Ozogul et al., 2010) reported that lipid oxidation of fatty acids can be reduced by adding natural and chemical antioxidants. Researchers added that vitamin-E found naturally in foods is available also as a dietary supplement. (Ohene-Adjei et al., 2004) stated that vitamin E is a group of fat-soluble compounds with signi icant antioxidant activities by inhibiting the production of reactive oxygen species which was produced during the oxidation of lipids.
Nanoparticles and nanotechnology are the recent method of choice involved in the processing, packaging, and ensuring the safety of seafood during storage. Nanoparticles in the form of metal oxides, salts, and composites conjugated with natural polymers or bioactive agents are used in seafood industries. Metal and metal oxide nanoparticle based packaging could be an effective material in terms of their antimicrobial potential. It helps to release antioxidant and antimicrobial compounds through the direct interaction with the food as well as helps to improve food stability (Dasgupta et al., 2015). Copper compounds (Cu) are one such metal which are hypotoxic and has a high antibacterial effect against bacterial species associated with ish spoilage (Raf i et al., 2010) . Copper nanoparticles have the potential to disrupt cell function and may reduce the ability of microorganisms to develop resistance against copper. Cu nanoparticles can penetrate the bacterial cell, damage the DNA, and release antimicrobial Cu + ions leading to cell death (Harikumar, 2016) .
Common hurdles in seafood preservations are salt, smoke, acids, temperature (high or low) and more recently redox potential (vacuum-packed products). In order to lower the preservative level, the hurdle technology concept has been developed. The process consists of using combined hurdles to establish an additive antimicrobial effect, inhibiting oxidative damages. This would in turn improve the safety and the sensory quality of food (Leroi, 2014) .
Based on this seafood preservation concept, the antibacterial and anti-oxidant potential of copper oxide nanoparticles coupled with vitamin-E (CuONP s +VE) was investigated by applying the novel hurdle factors against seafood pathogens and by studying the cell viability using MTT assays. The hurdle method is proposed to explain the signi icance of combined use of different preservation factors as synergistic effects instead of using a largeintensity preservation factor.
Hurdle technology method proposed in the present research work is considered as state of art of method by evaluating, the effect of CuONP s +VE and chilling temperatures (-18 • C and +4 • C); and the effect of CuONP s +VE and brine salts at two different concen-trations (5%, 10%) against total heterotrophic and total coliforms associated with the ish during storage conditions.

MATERIALS AND METHODS
The present research work was carried out in Department of Microbiology, Vivekanandha College of Arts and Science for women, Namakkal, Tamil Nadu, India. The research work was carried out during the period of November 2019 to February 2020. All chemicals and bacteriological media were procured from Hi Media Pvt Ltd (Mumbai, India).

Synthesis of copper oxide nanoparticles
Copper oxide nanoparticles were prepared using two prepared solutions separately. Solution -1: About 6.9g of copper sulphate pentahydarate was dissolved in 100ml of distilled water. To dissolve completely the mixture was stirred in a magnetic stirrer kept at constant conditions (37 • C and 200rpm). Solution-2: About 34.6g of sodium potassium taratate and 12g of sodium hydroxide was dissolved in 100ml of distilled water. To dissolve completely similar stirring conditions were carried out using a magnetic stirrer kept at constant conditions (37 • C and 200rpm). About 50ml of solution-1 and 50ml of solution-2 was mixed together with vigorous stirring and 5g of glucose was added and then the mixture was stirred vigorously was 10mins and then keep in boiling water bath at 60 • C for 10mins. Then, the obtained mixture is centrifuged and washed with distilled water twice and with ethanol twice and it was air dried and the powdered substance was used for further analysis (Kooti and Matouri, 2010) .

Preparation of Nanometal vitamin complex (CuONP s +VE)
A series of Nanometal vitamin complex were synthesized according to the following general procedure: copper oxide nanoparticles were added gradually to magnetically stirred ethanol solution (20ml). Then, to the irst reaction mixture, vitamin E oil was added and stirred carefully at about 60-80 • C till the reaction reached equilibrium. Then evaporation of the solvent (by placing the reaction mixture in a fume cupboard) and nanometal vitamin complex is synthesized (Fazary et al., 2016) .

Inhibitory effect of Nano-Metal Vitamin complex (CuONP s +VE) on the viable cells using MTT assay methods
The inhibitory effects of Nano-Metal Vitamin complex are determined on the viable bacterial cells (Escherichia coli Salmonella sp and Vibrio sp) using a standard MTT assay method. In brief, about 1ml of actively growing culture was taken and its absorbance indicating the prevalence of the organisms was calculated with help of UV-visible spectrophotometer at 600nm. At the development of cells on the culture a mixture of CuONPs+VE and MTT reagent is added and subjected to 10 minutes incubation. Centrifugation of the above sample at 8000rpm for 5mins resulted in crude pellet which was then redissolved in sterilized water.
The CFU was enumerated for both CuONPs+VE-MTT exposed and Control cells. The difference in CFU for test sample and control was calculated to express in percentage of cell inhibition (viable cells).

Cell inhibition (%) = A -B/A X 100
Where A -Control cells, and B -MTT exposed cells.

Identi ication of effective hurdle factors that could retard the growth of pathogens during different seafood storage conditions
To investigate the effect of CuONPs+VE along with other factors (chilling temperature, brine salt concentrations and pasteurization temperature) for the growth inhibition of THB (total heterotrophic bacteria) and TC (total coli irms) was studied. The factors that were selected for the inhibition of the organisms were thus referred to as Hurdle Factors. The effective hurdle factor that could inhibit the increase in number of bacterial gut microbiota in the stored seafood products (Fish) was evaluated in the present research work. Two different hurdle factors with their respective storage conditions were analysed.

Effect of CuONPs+VE + Chilling temperature ( • C)
About 400g of fresh marine ish was immersed in CuONPs+VE solution for 8h at room temperature (30 • C). The excess CuONPs+VE suspension was removed by manual gentle shaking for 30 seconds and packed in a sterile food packing kling ilms. A separate pack of ishes (100g) were stored at 4 • C in a refrigerator and the other pack of ishes (100g) was frozen at -18 • C (freezer). Similarly, another set of ishes without immersing in the CuONPs+VE (Control) were packed and stored at 4 • C and -18 • C respectively.

Effect of CuONPs+VE + Brine salt concentrations (%)
To detect the effect of brine salt as one of the hurdle factor, sodium chloride at four different concentrations (5%, 10%) was selected. Each concentration of salt solution was prepared and the marine ishes were immersed with and without CuONPs+VE suspension for 8h. Difference in the number of cells were enumerated as per microbiological standards and presented separately for total heterotrophic bacteria (THB) and total coliforms (TC).

Inhibitory effect of CuONPs+VE on the viable cells using MTT assay methods
The inhibitory effect of CuONPs+VE on the viable cells of food pathogens was evaluated using MTT assay methods.
In this method, the CFU of the control cells was compared with the CFU of the CuONPs+VE exposed cells. During the analysis, all the three pathogens exposed to MTT showed maximum cell inhibition Figure 1. The CuONPs+VE exposed Escherichia coli reduced upto 86±0.57% after enumerating the CFU in a Plate Count Agar media. Salmonella sp and Vibrio sp exhibited 89±1.04% and 85±0.25% respectively. The obtained results were found to be well in accordance to the results of MIC and antibacterial activity values; where the CuONPs+VE in the well diffusion method showed good inhibitory zones against all the three test organisms.
The cells were colorimetrically measured the by MTT assay, it is tetrazolium salt, which in the presence of fully functioned cell is reduced into a product possess for display of alive cells. The MTT assay reveals that the CuONPs+VE inhibited the metabolic activity of test organisms. The antimicrobial metal nanoparticles act on exterior region, tracked by lysis of cell there by reduction in biochemical reaction.
Identi ication of effective hurdle factors that could retard the growth of pathogens during different seafood storage conditions

Effect of CuONPs+VE + Chilling temperature ( • C)
The effect of CuONPs+VE and chilling temperatures for preservation of seafood products is evaluated

Total heterotrophic bacteria (THB)
The growth of heterotrophic bacteria (THB) at 4 • C was presented in Table 1 . At 1 st day, the control had 4x10 2 CFU per ml which was gradually increased upto 34 th day. On 34 th day, the growth was observed as 10x10 2 CFU/ml. About 6x10 2 CFU/ml were observed on 1 st week (7 th day). The cell count remained constant at 21 st and 28 th day (8x10 2 CFU/ml). Whereas, CuONPs+VE incorporated samples showed 3x10 2 CFU/ml in the 1 st day. The Total Heterotrophic Bacterial count (THB) increased to 3x10 2 CFU/ml at the14 th day. About 4x10 2 CFU/ml were observed at 34 th day. When compared to control signi icant heterotrophic bacterial growth difference was observed for CuONPs+VE exposed samples.
The growth of heterotrophic bacteria (THB) at -18 • C was presented in Table 2 . On 1 st and 7 th day, about 3x10 2 CFU per ml was observed for the control samples. The growth was slightly increased (4x10 2 CFU/ml) at 34 th day due to -18C. Samples in the presence of CuONPs+VE were found 1x10 2 CFU/ml in the 1 st day. The cell count remained constant at 7 th , 14 th and 21st day (2x10 2 CFU/ml). At 34 th day no CFU was evident, indicating that CuONPs+VE had signi icant inhibitory effect.

Total coliforms (TC)
The Total Coliform Growth (TC) on sample incorporated with CuONPs+VE and untreated at 4 • C was presented in Table 3. On 1 st day, the control had 3x10 2 CFU/ml. The bacterial cell count was increased at 7 th and 14 th day (4x10 2 CFU/ml) and started declining in the following weeks. At the 21 st day the total coliform count was 3x10 2 CFU/ml and the bacterial count remained constant till 28 th day (3x10 2 CFU/ml). About 2x10 2 CFU/ml was observed at 34 th day. Comparatively, the sample incorporated with CuONPs+VE had 2x10 2 CFU/ml at the 1 st and 7 th day and the cell count decreased on following days. About 1x10 2 CFU/ml growth was observed till 28 th day respectively. There was no cell growth observed at 34 th day. Table 4 represents the Total Coliform growth (TC) on control sample and sample incorporated with CuONPs+VE at -18 • C. On 1 st day, the control had 1x10 2 CFU/ml. The bacterial cell count remained constant till 14 th day. After 21 days, the total coliform count was 3x10 2 CFU/ml and CFU declined after 28 th day (2x10 2 CFU/ml). No cell growth was obtained at 34 th day. Comparatively, the sample incorporated with CuONPs+VE showed 1x10 2 CFU/ml from 1 st day to 21 st day. As coliforms are pathogenic bacteria which are found less in seafood, preserving under chilling temperatures incorporated with CuONPs+VE resulted in no growth from 28 th day. Due to the effect of low temperature, no signi icant difference in the control and nanoparticles exposed samples were evident. The results showed that hurdle factor did not played any role with the effect of -18 • C.

Effect of CuONPs+VE + Brine salt concentrations (%)
The effect of CuONPs+VE with brined samples was examined and optimization was done using different salt concentrations (5%, 10%, 15%, and 20%). The effect of CuONPs+VE is compared with the control i.e. the brined sample at different salt concentration. The results were observed weekly upto six weeks (34days). The growth of both heterotrophic bacteria and coliforms are evaluated and tabulated. Table 5 represents the growth of heterotrophic bacteria (THB) at 5% salt concentration. On 1 st day, the control had 7x10 2 CFU per ml which was gradually decreased upto 21 th day. The growth was observed as 5x10 2 CFU/ml at 34 th day. About 4x10 2 CFU/ml was observed on 7 th day for CuONPs+VE exposed samples. The cell count remained constant at 14 th and 21 st day (4x10 2 CFU/ml). About 3x10 2 CFU/ml were present at 28 th day respectively. The Total    Heterotrophic Bacterial count (THB) decreased to 2x10 2 CFU/ml at 34 th day which is signi icantly decreased when compared to that of control samples. The hurdle factor played an important role when the nanoparticles are coupled with 5% salt concentrations.

Total heterotrophic bacteria (THB)
From the above results it is clear that, increase in salt concentration increases the inhibitory effect. Moreover, the rate of inhibition of THB was higher in CuONPs+VE incorporated samples than the plain brined samples and the effective concentration was found to be 20%.
The growth of heterotrophic bacteria (THB) at 10% salt concentration is presented in Table 6 . On 1 st day, the control had 6x10 2 CFU per ml which was found to be increased in 7 th day (6x10 2 CFU per ml). The growth was observed as 5x10 2 CFU/ml at 21 st day and 4x10 2 CFU/ml at 28 th day. About 5x10 2 CFU/ml was observed at 34 th day respectively. Whereas in the presence of CuONPs+VE there were 2 x10 2 CFU/ml present in the 1 st day. The heterotrophic bacterial count increased to 4x10 2 CFU/ml at 7 th and 4 th day. About 3x10 2 CFU/ml was observed at 21 st day and 1x10 2 CFU/ml were present at 28 th day. On 34 th day there cell growth remained constant as they prevented the growth of heterotrophic bacteria. Table 7 represents the growth of Total Coliforms (TC) at 5% salt concentration. On 1 st day, the control had 5x10 2 CFU per ml which was gradually increased at 7 th day (7x10 2 CFU/ml). The growth was observed as 6x10 2 CFU/ml at 14 th day. About 5x10 2 CFU/ml was observed on 28 th day. The cell count remained constant at 28 th day and 34 th day (4x10 2 CFU/ml) respectively. Whereas, CuONPs+VE incorporated samples showed 4x10 2 CFU/ml in the   CFU/ml at 14 th day. The TC remained constant at 21 st at 28 th day (3x10 2 CFU/ml) and about 4x10 2 CFU/ml was observed at 34 th day respectively.
The growth of Total Coliforms (TC) at 10% salt concentration is presented in Table 8. On 1 st day, the control had 15x10 2 CFU per ml which was found to be increased in the 7 th day (6x10 2 CFU per ml). The growth was observed as 5x10 2 CFU/ml at 21 st day and 4x10 2 CFU/ml at 28 th day. The growth was decreased and about 3x10 2 CFU/ml was observed at 34 th day respectively. Whereas in the presence of CuONPs+VE there were 3 x10 2 CFU/ml present in the 1 st day. The bacterial count was 3x10 2 CFU/ml at 7 th day and decreased to 2x10 2 CFU/ml at 14 th day. About 1x10 2 CFU/ml was observed at 21 st day and 2x10 2 CFU/ml at 28 th day. On 34 th day the total number of coliforms was found to be 1x10 2 CFU/ml. From the above results it is clear that, the rate of inhibition of TC was higher in CuONPs+VE incorporated samples than the plain brined samples and the effective concentration was found to be 20%.
Salting is one of the oldest food preservation methods. Salting of seafood is done with salt. Chlorine and sodium ions are carried from brine to ish, and water dipoles are carried from ish to the environment. The effects of brine and dry salting methods on the nutritional composition of chub and the changes on microbial that arise during storage were investigated by (Thorarinsdottir et al., 2004). It was determined that crude protein, lipid, crude ash and salt amounts in the group where dry salting method was applied were higher than the group where brine salting occurred, in addition protein and lipid values decreased as storage period was longer. It was determined that there is an increase in total aerobic mesophilic, psycrophile bacteria and enumeration of yeast and mould as storage period increased, while coliform bacteria decreased.
The Effect of CuONPs+VE and chilling temperatures (-18 • C and +4 • C) and effects of CuONPs+VE and brine salts at different concentration (5%, 10%, 15%, 20%) were studied. CuONPs+VE with different chilling temperatures showed signi icant results on compared to control temperatures. Similarly CuONPs+VE with brine salts at different concentration showed better results than the control sample containing brine salts.

CONCLUSIONS
Effect of CuONPs+VE and chilling temperature (-18 • C and +4 • C); and Effect of CuONPs+VE and brine salts at various concentrations (5%, 10%) were evaluated. CuONPs+VE with different chilling temperatures and brine salt concentration showed signi icant results on compared to control temperatures. Thus CuONPs+VE due to their bacteriostatic activity can be ef iciently used in hurdle technology which reduces the food spoiling organisms. Thus CuONPs+VE in combined with hurdle technology can be used as alternatives for chemical preservatives in preservation techniques. The suggested process would address antimicrobial activity as well as destructive oxidation of the desired lipids and fats. However, more efforts are required to understand the role of proximate composition of ish, postharvest history, environmental conditions, initial microbial load, type and nature of bacteria and their interaction in order to optimize the shelf-life of ish.