Development and evaluation of a vacuum impregnation system for enhancement of biochemical properties of food materials

Abstract

Vacuum impregnation is a novel methodology for adding various substances to porous foods. This study aimed to develop a cost effective automate system for vacuum impregnation of food materials to enhance their nutritional, functional and sensory properties depending on the functionality of the impregnation solution. The developed vacuum impregnation system includes a vacuum chamber, vacuum pump and an automation setup for creating and maintaining vacuum conditions, feeding impregnated solutions to the samples and releasing vacuum. Fresh-cut spinach leaves were impregnated with ascorbic acid (AsA) and calcium chloride (Cacl₂) (10% concentration) in the setup in order to test the effect of the process on some biochemical properties. Statistical analysis revealed significant effect of vacuum impregnation on the biochemical properties (total soluble solids, total phenolic content, flavonoid content and free radical scavenging activity) and color of spinach leaves during storage up to 4 days. Impregnation process showed significant increase in the total phenolic and flavonoid content of the spinach leaves. Increment up to 78% in antioxidant activity was seen for the uncoated impregnated leaves as compared to 59% activity in untreated samples. Thus, products with desired parameters can be produced with this process with minimal impact on their properties at a lower cost and in a shorter time period.

Introduction

Food quality is becoming increasingly important to consumers, and they expect the quality of their food to remain high during the interval between purchase and consumption. Beyond the fundamental concern for upholding food safety standards, this escalating expectation is underpinned by an additional imperative: the necessity to ensure that the sensory characteristics of food items do not incur any detrimental impacts. With fresh produce, this becomes even more important. Fresh produce has a shorter shelf life and contributes significantly to post-harvest losses. The fact that each vegetable is unique sets fresh produce apart from other food products. The manner in which a produce behaves is influenced both by its genetic make-up, its stage of development, and the circumstances it faced prior to and following harvest. Fresh produce has a much more complex composition and stability than products with a more undeviating composition, which makes shelf-life prediction particularly challenging. IFT (1974) delineation of shelf-life, as: “The period between manufacture and retail purchase of a food product during which the product is of satisfactory quality”.

Leafy green vegetables have a high nutritional content, low calorie content and prophylactic properties, due to which they have become part of modern diet. Regardless, raw materials from these plants may become contaminated with high amounts of microorganisms and even pathogens as a result of their amalgamation with soil and post-harvest processing, resulting in foodborne outbreaks (Jo and Park 2019). As a result, enhanced nutritional and functional benefits are now the focus of improvement. Basically, process improvement is focused on making the product as nutritious as possible and safe for consumption by optimizing the resources used, so that their nutritional and beneficial characteristics are maintained during post-harvest handling and storage. Technologists, engineers, and companies are challenged in this direction to develop new techniques for food processing (Buckle 2001). Traditional processing technologies have a detrimental influence on functionality of fresh produces in general, but recent research has demonstrated that processing technologies can be effectively managed to lessen impact and even improve functionality. One of the novel approaches to food preservation/fortification is vacuum impregnation (VI), which is distinguished by its peculiar effect on the structure of food, its active compounds, versatility, and their wide application alone or in combination.

Impregnation under vacuum is accomplished by exchanging the gas and native liquid with an impregnation solution that results in a pressure change (Castagnini et al. 2015). Capillary pressure at the pores’ ingress generates pressure gradients, which greatly facilitate gas and liquid transfer between the solid and liquid. Researchers (Fito 1994; Fito and Pastor 1994) explained VI operation as a hydrodynamic phenomenon built on the porous structure of various plant tissues and the presence of gas obscured there. The occluded gas in the solid is expanded by sub-atmospheric pressures when it is immersed in a liquid where there is sub-atmospheric pressure. Depending on the applied pressure, this results in structural degasification of the pores, and at equilibrium, liquid is able to penetrate the porous structure via capillary action. In addition, the refurbishment of atmospheric pressure in the structure dictates produce a new pressure gradient which acts as an influential force, and lets the product’s intercellular spaces to partially fill with external liquid. In addition to degasification level, pressure will also affect the amount of liquid that impregnates the solid structure. In general, the quality improvement of foods through VI pre-treatment is a gentle treatment of the product at ambient temperature, which allows natural flavor, color, aroma, and heat-receptive nutrients to remain intact. Technology such as this is widely used in pre-treatment prior to drying, freezing, and frying (Bolin and Huxsoll 1993). Alzamora et al. (2000) reported that oxygen removal from the pores during VI inhibits enzymatic and oxidative browning, thus preventing fruit discoloration. Utilizing vacuum impregnation during fresh produce processing has numerous improvements such as mineral and vitamin enhancement as such or before drying, freezing, and canning, modification of product formulations, and many others (Fito et al. 2001; Zhao and Xie 2004).

Spinach is a prevalent dark green leafy vegetable for its rich in folate, carotene (provitamin A), calcium, phosphorus, iron, potassium, sodium and as well as in vitamin C (Zhao and Xie 2004). The pigment lutein, that has been found to aid in preventing age-related macular degeneration, is abundant in this food. Spinach is strong in antioxidants and has one of the maximum oxygen radical absorbance capacity ratings of any vegetable (Ryder 1979).

Through vacuum impregnation (VI), the purpose of the research presented in this paper was to determine if fresh produce such as spinach can be enriched with functional compounds. An automated VI system was developed for this purpose to study the effect of impregnation on some biochemical quality parameters of the fresh leafy vegetable. Using the cost economics analysis, it is possible to better assess the proposed technology in terms of sustainability, environmental impact, and economic value.

Materials and methods

Raw materials

The spinach (Spinacia oleracea L.) leaves used in the experiments were obtained from the Farm Section of ICAR-Central Institute of Agricultural Engineering, Bhopal, India (23.3156° N, 77.4037° E). The leaves were thoroughly washed in running tap water before being used in the experiment. The leaves were used in two ways for the experiment: directly (uncoated) and coated with beeswax at the tip of the leaf. Coating was done to test the impregnation effect.

Development of a vacuum impregnation system

The developed vacuum impregnation system includes a vacuum chamber, vacuum pump and an automation setup. The actual set-up is depicted in Fig. 1a with its CAD model (Fig. 1b). The vacuum chamber with a dimension of 260 (L) × 260 (W) × 280 (H) mm and made up of 25 mm thick acrylic sheet was developed. The chamber dimensions were designed so as to hold the sample vessel and create the necessary vacuum environment. A high-performance single stage vacuum pump of capacity 51 LPM, RPM of 2880, power of 150W and frequency of 220 V/50HZ was used to create the required vacuum in the chamber.

Fig. 1.

The Vacuum impregnation system

The automation setup included a vacuum sensor of range −1 to + 5 Bar; a temperature sensor (Pt100) of range 0 to 200 °C with a precision of 0.1 °C; a digital display unit (4 digit) with dimension of 96(W) × 48(H) × 77(L) mm; a programmable logic controller (PLC) of accuracy of 0.25% ± 1 °C (TC), 0.1% ± 1 °C resistance temperature detector(RTD) at 25 C ± 0.25% of full scale, ± 1 count for AIs (Analog inputs) Conversion time 100 ms; and a Display 7 Segment LED Digits (4 + 4) Red + 6 Green Digits LED Bank 4 RED LED’s, 4 GREEN LED’s with 5 Keys (4 User Configurable). The display unit was used to provide information about the various stages of process and also used for feeding inputs.

The sensors, contractor, and PLC that were selected determined the system’s automation. As a result, the sensor’s precision was demonstrated in all atmospheric phenomena, allowing the system’s timing and fusion to be evaluated. The sensors, contractor, and relay module were linked using the electrical circuit and arrangement designed for laboratory conditions. The flow chart of the entire system is shown in Fig. 2a, displaying all the connections throughout the system.

Fig. 2.

a Flowchart of the impregnation automation setup; b Phases of Vacuum Impregnation process

Evaluation of the vacuum impregnation system

Experiments were conducted for vacuum impregnation of spinach leaves in the specified system with solutions of ascorbic acid (AsA) and calcium chloride (CaCl₂) mixtures (1:1 ratio, 10 percent concentration). The leaf samples were kept in the sample vessel and put within the vacuum chamber following its preliminary weight perseverance. The vacuum process was started until the required vacuum of 450.03 mm Hg was achieved. The leaves were immersed with the liquid after achieving vacuum and held under vacuum for 10 min. Following the vacuum phase, air pressure was restored, and the samples were kept immersed for 15 min to complete the restoration process. Impregnated samples after surface drying were packed in LPDE pouches with thickness of 80 μm and stored at 25 °C.

Quality evaluation of impregnated samples

Vacuum pressure, vacuum time, restoration time and concentration of solution are various parameters that determine the successful impregnation of the solution. To study the performance of the VI system and effect of these parameters on product quality, the following analysis were performed during the storage period which were compared with the fresh stored samples as well.

Determination of biochemical parameters of spinach leaves

Total phenolic content (TPC), flavonoid content (FC), ascorbic acid content (AsA), free radical scavenging activity (FRAS) and total soluble solid (TSS) of impregnated leaves were determined. Three replicates of each sample were examined for each parameter.

The total soluble solids (TSS) of spinach leaves were measured at a temperature of 20 °C using a handheld pocket refractometer (PAL-1, ATAGO, Japan). The Folin-Ciocalteu reagent was used to compute total phenols in accordance with the modified procedure by Singleton and Lamuela (1999). Initially, 0.5 g of fresh broccoli florets were combined with an 80% methanolic solution and subjected to centrifugation at 553.41 G for 20 minutes using a C 24 Remi Group Laboratory Instruments centrifuge (India). The resulting supernatant was obtained by filtering the methanolic extract through qualitative filter paper (100125 R, AXIVA SICHEM BIOTECH, India). Subsequently, 0.2 mL of the methanolic extract was mixed with 10 mL of Foilin–Ciocalteu reagent (diluted 10-fold), followed by adding 8 mL of 7.5% sodium carbonate. After incubating in darkness for 90 min, the absorbance at 765 nm was measured. A gallic acid equivalent (GAE) per 100 g of fresh weight was calculated based on the obtained values. A spectrophotometric procedure was used for the quantification of total flavonoids in spinach by aluminum chloride procedure by Zhishen et al. (1999). The TFC of florets was determined at 510 nm using the same extraction as that of phenolic content. Ascorbic acid was determined by spectrophotometric method using Folin-Ciocalteu reagent (FCR) at 760 nm as described by Rajoriya et al. (2019). The Spectrophotometric approach described by Erken & Kaya (2017) was used to determine antioxidant activity at 517 nm. Calculating the following equation, DPPH scavenging activity of the samples was governed as a reduction in absorbance:

$$\%\text{inhibition}=\frac{\text{Control}\text{OD}-\text{Sample}\text{OD}}{\text{Control}\text{OD}}\ast 100$$

Measurement of color

A computer vision-based image analysis method (Sabat et al. 2021) was used to measure the colors of fruits and vegetables (MATLAB, version R2020a, MathWorks, Natick, MA). The images were captured under uniform environmental conditions with constant lightning illumination (D65 illumination) with a digital camera. Pictures were captured in jpeg format on a personal computer without compression. The region of interest in the digital image was utilized to use a built-in MATLAB method to generate the L, a and b values from RGB values. Additionally, L, a, and b values were employed to designate color changes during storage. These values were total color change (ΔE), hue angle (h*), chroma and browning index (BI) calculated by the following equations.

$$\mathrm{\Delta }\text{E}=\sqrt{{(L_0-L_t)}^2+{(a_o-a_t)}^2+{(b_o-b_t)}^2}$$

$$\text{h}\ast ={\text{tan}}^{-1}\left(\frac{b_t}{a_t}\right)$$

$$\text{Chroma}=\sqrt{a_t^2-b_t^2}$$

$$\text{BI}=\frac{\left[100\left(x-0.31\right)\right]}{0.17};\text{where}x=\frac{\left(a_t+1.75L_t\right)}{\left(5.645L_t+a_t-3.012b_t\right)}$$

where L₀, a₀, b₀ are the initial color values of the samples, and L_t, a_t, b_t are the color measurements after impregnation treatment.

Since the h* considers both a_t and b_t values, it might be more representative of color than a single-color property like L_t (Demirhan and Ozbek 2009).

Statistical analysis

Statistical analysis was carried out for comparison of means of biochemical and color parameters at different treatments and storage periods. SPSS 20.0 (SPSS Inc., Chicago, USA) was used for the analysis of variance. The mean values of different parameters were compared by Tukey’s (b) test at 95% confidence interval.

Cost economic analysis of the process

The assessment of the cost economics for the vacuum impregnation process involved the expenses related to materials needed for fabrication and overhead costs. Both fixed and variable expenses were considered while determining the cost of the developed system. The following components were examined for fixed and variable costs, with details provided below:

a) Fixed cost = $i{(i+1)}^N/{(i+1)}^N-1\ast C$

where: i = Rate of interest, 10%

N = Lifecycle, years.

C = Cost of unit, Rs.

Housing insurance and taxes at the rate of 3% of initial cost.

b) Variable cost.

(i) Annual use, 200 days.

(ii) Repair and maintenance @ 2% of initial cost.

(iii) Electricity charges, Rs 7 per unit.

Result and discussion

Working process of the system

The vacuum impregnation involves several phases, which are exemplified in Fig. 2b. As the system was designed to be automated, only few inputs were required for the system to run smoothly using the keys on the digital display. Once the system is switched on, the first step is to enter the required vacuum level, which is represented as UAC on the digital display. After entering the vacuum level, the next step is to enter the vacuum time, it’s the time taken to achieve the required level. This step was achieved by calibrating the time-pressure relationship comparing both digital and by analog value of pressure on the pressure gauge installed on the system.

UON represents the dry cycle time (vacuum). The solution valve opens for the necessary time period shown as UOU on the display once the required vacuum level is reached. The impregnation time of the sample immersed in as is shown as UUF the wet cycle timing. The following phase is exhaust, which involves returning to air pressure after achieving the desired impregnation period. This phase is achieved by entering the required exhaust time or pushing the exhaust push button. After completing all of the vacuum impregnation cycles, the final step is to achieve the restoration time, which is amount of time the sample is immersed in the impregnation solution at atmospheric pressure. This is one of the utmost significant stages in the course as the voids formed during the vacuum cycle are filled by impregnation solution in this step, and new microscopic structures are formed. The developed system took minimum of 12 min to complete one cycle of process.

Biochemical properties of impregnated spinach leaves

Impregnation process has shown changes on the biochemicals properties of the spinach leaves. As shown in Fig. 3a, untreated and treated (coated and uncoated) leaves differ in the levels of biochemical content. TPCs of fresh spinach leaves were found to be 108 mg GAE/100 g FW. This value was found closer to that measured in spinach as 104.7 mg GAE/100 g FW by Mazzucotelli et al. (2018). However, the TPC value was found lower compared to that of 182.5 mg GAE/100 g FW (Bayili et al. 2011), but found to be higher compared to the value reported by Sun et al. (2002) as 90 mg GAE/100 g FW. Variety of spinach, harvest ripeness, and growth conditions are just a few factors of the many variables that could affect TPC levels (Howard et al. 2002).

Fig. 3.

Effect of storage days on (a) biochemical compounds; (b) TSS and (C) color parameters of treated and untreated spinach leaves (TPC- Total Phenolic Content, FC- Flavonoid Content, AsA- Ascorbic Acid Content, FRAS- Free Radical Scavenging Activity, TSS- Total Soluble Solids)

After impregnation of spinach leaves the TPCs were found to have increased to 220 mg GAE/100 g FW and 185 mg GAE/100 g FW in uncoated and coated spinach leaves, respectively. Flavonoid content in fresh leaves was found to be 145 mg QE/100 g FW, which was close to the value as reported by Mazzucotelli et al. (2018) i.e., 160.6 mg QE/100 g FW. After impregnation, the flavonoid content increased in both coated and uncoated spinach leaves to 176 mg QE/100 g FW and 181 mg QE/100 g FW, respectively. Ascorbic acid content in fresh leaves was found to be 78.23 mg/100 g which was higher than analyzed previously in literature as 60.6 mg/100 g (Yadav et al. 2013). Impregnation increased the ascorbic acid content as the impregnation solution itself contained ascorbic acid. The increment was observed to be more than twice the initial content for uncoated leaves (175.58 mg/100 g), whereas for coated leaves it increased up to 137.67 mg/100 g. The antioxidant scavenging activity of fresh leaves was found to be 59.06% which was in close agreement with activity i.e., 55.07% as quoted in literature (Kaliyaperumal & Radhika 2020). Impregnation process showed an increment in the antioxidant activity of the spinach leaves the increment was higher for the uncoated leaves than the coated. Increment of 78% activity was seen for the uncoated leaves and 65% for the coated leaves. The increase in the biochemical properties may have increased due the hydrodynamic mechanism (HDM), and simultaneously deformation-relaxation phenomena. The permeability of plant tissue increases mass transfer, therefore it’s feasible that VI treatment could accelerate VI processing. The total soluble solids (TSS) of the leaves increased from 3.3°Bx in untreated leaves to 3.9°Bx in case of uncoated and 3.4°Bx in coated and vacuum impregnated leaves (Fig. 3b).

Effect of storage period on biochemical properties

The values of different biochemical parameters decreased with storage duration, which proved the adverse effect of storage temperature along with storage period on the bioactive compounds. Statistical analysis revealed the significant effect of storage period (days) on biochemical compounds (p ≤ 0.05) (Table 1). The results depicted that some compounds exhibited a pronounced dilapidation rate over others when subjected to storage at ambient temperatures. When compared with treated samples, the untreated samples demonstrated a higher decline in the biochemical parameters. Such effect occurs often due to oxidation reaction, along with quenching of free radicals by polyphenol substances. The influence of change in content were seen minimum in the impregnated samples due to higher level of AsA after impregnation (Moreira & Almohaimeed 2018). Notably, the uncoated impregnated samples showed the lowest degree of deterioration, likely attributed to their comparatively higher level of impregnation compared to the coated leaves. The loss of carbohydrates, chlorophyll, vitamins, and acids, as well as a higher respiration rate, are the primary causes of the decrease of biochemical compounds in spinach leaves during storage. Tissue dehydration occurs as the produce loses water during storage, resulting in changes in texture and structure that can affect firmness of the produce (Toivonen & Brummell 2008).

Table 1.

Effect of vacuum impregnation on biochemical parameters of coated and uncoated spinach leaves during storage

Biochemical properties	Treatments	Storage days
		1	2	3	4
TPC (mg GAE/100 g FW)	Untreated	108 ± 0.82aA	96 ± 0.82aB	83 ± 0.82aC	69 ± 0.82aD
	Treated (Coated)	185 ± 0.82bA	184 ± 0.79bB	175 ± 0.81bC	166 ± 0.89bD
	Treated (Uncoated)	220 ± 0.81cA	214.3 ± 0.47cB	208 ± 1.14cC	200 ± 1.39cD
FC (mg QE/100 g FW)	Untreated	145 ± 1.52aA	90 ± 0.72aB	81 ± 1.12aC	73 ± 0.49aD
	Treated (Coated)	176 ± 0.95bA	169 ± 0.45bB	155 ± 0.87bC	143 ± 0.73bD
	Treated (Uncoated)	181 ± 0.92cA	174 ± 0.44cB	166 ± 0.97cC	158 ± 0.80cD
AsA (mg/100 g)	Untreated	78 ± 0.21aA	66 ± 0.59aB	53 ± 0.42aC	49 ± 0.66aD
	Treated (Coated)	137 ± 1.15bB	129 ± 1.47bB	125 ± 1.36bC	116 ± 0.99bD
	Treated (Uncoated)	175 ± 0.45cC	170 ± 0.49cB	164 ± 0.82cC	160 ± 1.25cD
FRSA (%)	Untreated	59.09 ± 0.83aA	52 ± 0.56aB	47 ± 0.56aC	38 ± 1.02aD
	Treated (Coated)	65.57 ± 1.45bA	62 ± 1.03bB	59 ± 0.13bC	48 ± 0.3bD
	Treated (Uncoated)	78.38 ± 0.23cA	73 ± 0.82cB	69 ± 0.51cC	65 ± 0.56cD
TSS (°Bx)	Untreated	3.3 ± 0.08aA	3.1 ± 0.16aA	3 ± 0.08aC	2.8 ± 0.17ac
	Treated (Coated)	3.4 ± 0.22aA	3.3 ± 0.14bB	3.1 ± 0.17bC	3 ± 0.14bD
	Treated (Uncoated)	3.9 ± 0.08cA	3.8 ± 0.16cB	3.6 ± 0.08cC	3.6 ± 0.14cD

Means followed by the upper-case letters on column (Storage days), and means followed by the lower-case letters row (Treatments) differ significantly by the Tukey test (p ≤ 0.05). Values are mean of 3 replications

TPC- Total Phenolic Content, FC- Flavonoid Content, AsA- Ascorbic Acid Content, FRAS- Free Radical Scavenging Activity, TSS- Total Soluble Solids

Zhang et al. (2022) observed certain correlation between the biochemical reactions occurring in fresh cut vegetables during storage. The correlation analysis of spinach leaves impregnated with AsA and CACl₂ untreated throughout storage is shown in Table 2. The AsA content showed positive significant correlation with TPC, FC (P < 0.05) and was negatively correlated with TSS, while it has a non- significant correlation with FRSA. Due to their fragile structure under numerous conditions surrounding the sample such as light, pH, storage temperature, and time, the phenolic compounds are easily susceptible to deterioration. As temperature rises, phenolic compounds may degrade, rapidly reducing their antioxidant activity in food (Kim & Padilla-Zakour 2004). The deterioration in the bio-compound associated with the oxidation reactions (enzymatic activities) which leads to the interruption of plant tissue in the membranes of cells. Assortment of cell starts to fail once a stress or spoilage process is initiated (Toivonen & Brummell 2008; Marangoni et al. 1996).

Table 2.

Correlation study of spinach leaf components throughout storage period

	TPC	FC	AsA	FRSA	TSS
TPC	1*
FC	0.84*	1
AsA	0.94*	0.89*	1
FRSA	−0.19**	−0.06**	−0.16**	1
TSS	−0.53*	−0.39*	−0.49*	0.15**	1

^*Significant correlation level (P < 0.05), **non-significant correlation

TPC- Total Phenolic Content, FC- Flavonoid Content, AsA- Ascorbic Acid Content, FRAS- Free Radical Scavenging Activity, TSS- Total Soluble Solids

Color parameters

The ‘L’ and’b’ values in untreated spinach progressively declined along storage whereas ‘a’ value increased, which specified that color of spinach leaves has develop to be less green and darker. In contrast, impregnation efficiently prevented the decline in L, a and b values. It was observed that in case of untreated spinach, there was 44.98% and 29.85% decrease in the ‘L’ and ‘b’ values, respectively and 18.96% increase in ‘a’ values from the initial values of the parameters after four days of storage. In contrary, a decrease of 27.14 and 6.48% in ‘L’ values; 14.27 and 3.73% in ‘b’ values and an increase of 9.39 and 3.97% in ‘a’ values were noticed for impregnated treated coated and treated uncoated samples, respectively. Statistical analysis revealed that spinach leaves had significant differences in L, a and b values for treated (both coated and uncoated) versus untreated samples during storage period (p ≤ 0.05) (Table 3). Results also indicated the increase in hue, browning index and decrease in ΔE with maximum changes for untreated leaves followed by treated coated and treated uncoated samples (Table 3 and Fig. 3c).

Table 3.

Changes in color parameters of vacuum impregnated and untreated spinach leaves over storage period

Treatments	Storage period (Days)	Color parameters
		L*	a*	b*	ΔE	hue	chroma	BI
Untreated	1	11.07 ± 0.25aA	−24.79 ± 0.12bA	17.42 ± 0.13cA	11.07 ± 0.11dA	180.65 ± 0.00eA	25.23 ± 0.16fA	33.32 ± 4.58gA
	2	9.69 ± 0.11aB	−23.46 ± 0.11bB	16.03 ± 0.10cB	9.69 ± 0.17 dB	180.66 ± 0.00eB	24.39 ± 0.15fB	41.89 ± 2.75gB
	3	7.78 ± 0.20aC	−21.55 ± 0.15bC	14.08 ± 0.10cC	7.78 ± 0.21dC	180.67 ± 0.00eC	22.72 ± 0.18fC	50.59 ± 2.83gC
	4	6.08 ± 0.14aD	−20.09 ± 0.22bD	12.22 ± 0.09cD	6.08 ± 0.26dD	180.70 ± 0.00eD	21.36 ± 0.23fD	64.00 ± 2.56gD
Treated (Coated)	1	12.50 ± 0.36aA	−18.79 ± 0.11bA	14.36 ± 0.14cA	12.50 ± 0.21dA	179.35 ± 0.01eA	23.65 ± 0.05fA	30.64 ± 3.46gA
	2	11.87 ± 0.09bB	−18.05 ± 0.53bB	13.82 ± 0.22cB	11.87 ± 0.30 dB	179.34 ± 0.02eB	22.74 ± 0.30fB	35.86 ± 6.72gB
	3	10.95 ±  ± 0.26cC	−17.23 ± 0.12 bC	13.13 ± 0.04cC	10.95 ± 0.19dC	179.33 ± 0.00eC	21.67 ± 0.12fC	43.17 ± 5.65gC
	4	9.10 ±  ± 0.03dD	−17.02 ± 0.19bD	12.31 ± 0.07cD	9.10 ± 0.12dD	179.35 ± 0.01eD	21.00 ± 0.12fD	55.01 ± 5.46gD
Treated (Uncoated)	1	11.67 ± 0.18aA	−18.26 ± 0.13bA	13.84 ± 0.14cA	11.67 ± 0.23dA	179.35 ± 0.00eA	22.92 ± 0.17fA	32.95 ± 1.32gA
	2	12.74 ± 0.17aB	−18.37 ± 0.08bB	14.26 ± 0.12cB	12.74 ± 0.19dB	179.34 ± 0.00eB	23.26 ± 0.13fB	35.48 ± 1.59gB
	3	11.30 ± 0.31cC	−17.83 ± 0.30bC	13.57 ± 0.10cC	11.30 ± 0.23dC	179.35 ± 0.01eC	22.41 ± 0.24fC	42.46 ± 5.09gC
	4	10.91 ± 0.16dD	−17.53 ± 0.20bD	13.33 ± 0.08cD	10.91 ± 0.12dD	179.35 ± 0.00eD	22.02 ± 0.21fD	52.56 ± 9.09gD

Means followed by the upper-case letters on the same column (Color parameters) and means followed by the lower-case letters on the same row (Days) differ significantly by the Tukey test (p ≤ 0.05). Values are mean of 3 replications. Means with the same letter are not significantly different in a column at 95% confidence interval. L*- Lightness, a*- Red-Green. b*- Yellow-Blue, ΔE—total color change, BI—Browning index

Results of our study divulged that impregnation might effectually uphold the lightness of specious color in spinach, along with browning inhibition, thus, keeping higher rate of marketable acceptability. Enzymatic browning occurs due to the reactions between oxidative enzymes and phenolic compounds resulting to cellular interruption (Massolo et al. 2011).

It’s important to be aware of the potential consequences of surface cell rupture and stress on underlying tissues. One such consequence is increased enzymatic activity, which can lead to cell membrane permeability and mixing of enzymes and substrates that are typically isolated in vacuoles. Interaction of polyphenol oxidase in the presence of oxygen with substrate of polyphenolic compound results in browning. Catalysis reaction of polyphenol oxidase are of two types. Firstly, the hydroxylation of monophenols to diphenols. Other is the formation of quinones by oxidation of diphenol compounds. In our study oxidation reaction may have occurred due to the resultant quinones. Consequent responses of the quinones might have led to accumulation of melanin pigment (brown or black) linked to plant tissue browning depending on the polyphenolic substrate. The results suggested brown color of spinach leaves in the untreated samples, but it was not very noticeable in the impregnated samples and the increase in BI was minimum.

However, tissue injury can promote respiration and ethylene production. To slow down ethylene synthesis and respiration, substances such as ascorbic acid and calcium chloride can be used. These substances can help to keep cells from degrading. The inhibition of browning by calcium chloride and ascorbic acid be due to inhibition of polyphenol oxidase and peroxidase activity of phenolic compounds to form quinone compounds by the chloride ion and ascorbate is been reported in literature (Apintanapong et al. 2007; Duan et al. 2009). These results were also backed-up by the maintenance of total phenolic and soluble solid content as well as the integrity of the cell membrane due to impregnation during storage.

Cost economics for vacuum impregnation process

The cost of the developed system (C) was calculated to be INR 59354.00

$$\text{Capacity}\text{of}\text{system}=\text{2 kg}/\text{h}$$

$$\text{Life}\text{span}\left(\text{N}\right)=\text{5 years}$$

$$\text{Working}\text{hours}=\text{8 h}/\text{day}$$

$$\text{Annual}\text{usage}=200\text{days}$$

$$\text{Electricity}\text{charges}=\text{INR 7}/\text{kWh}$$

Fixed Cost/year:

Interest rate (i) = 10%

$$\begin{array}{cc}\hfill \text{Fixed}\text{cost}\text{of}\text{system}\left(\text{I}\right)=& i{\left(i+1\right)}^N/{\left(i+1\right)}^N-1\ast C\hfill \\ \hfill & =0.1{\left(0.1+1\right)}^5/{\left(0.1+1\right)}^5-1\ast 59354\hfill \\ \hfill & =\text{INR}15,657.00\hfill \\ \hfill \end{array}$$

$$\begin{array}{cc}\hfill \text{Insurance}\text{and}\text{taxes}\left(\text{II}\right)=& 3\%\text{of}\text{initial}\text{cost}\text{of}\text{unit}\hfill \\ \hfill & =0.03\ast 59354\hfill \\ \hfill & \text{= INR 1781}.00\hfill \\ \hfill \end{array}$$

$$\text{Total}\text{fixed}\text{cost}/\text{year}=\left(\text{I}\right)+\left(\text{II}\right)=\text{INR 17},438.00$$

$$\text{Total}\text{fixed}\text{cost}/\text{hr}\left(\text{a}\right)=\text{Rs 17},438/1600=\text{INR 1}0.90$$

Variable cost/ year

$$\begin{array}{cc}\hfill \left(\text{I}\right)\text{Repair}\text{and}\text{maintenance}=& 2\%\text{of}\text{initial}\text{cost}\text{of}\text{unit}\hfill \\ \hfill & =0.02\times 59354=\text{INR1186}.90/\text{year}\hfill \\ \hfill \end{array}$$

$$\begin{array}{cc}\hfill \left(\text{II}\right)\text{Electricity}\text{charges}=& \text{power}\text{source}1\text{hp}=0.746\text{kWh}\hfill \\ \hfill & =\text{INR 7}/\text{kWh}\times 0.\text{746 kW}\hfill \\ \hfill & =\text{INR 5}.23/\text{h}\hfill \\ \hfill & =5.23\ast 1600=\text{INR 8368}/\text{year}\hfill \\ \hfill \end{array}$$

$$\left(\text{III}\right)\text{Chemical}\text{and}\text{other}=\text{INR}3000/\text{year}$$

$$\text{Total}\text{variable}\text{cost}/\text{year}=\left(\text{i}\right)+\left(\text{ii}\right)+\left(\text{iii}\right)=\text{INR12},554.90$$

$$\text{Total variable cost}/\text{h}\left(\text{b}\right)=\text{INR 7}.85$$

$$\text{Average}\text{production}/h=\text{2 kg}$$

$$\text{Total}\text{production}/\text{day}=2\times 5=10\text{kg}$$

$$\text{Cost}\text{of}\text{operation}\text{of}\text{machine}/\text{h}\text{= a}+\text{b}=17.44+7.85=\text{INR 25}.29$$

$$\text{Cost}\text{of}\text{treatment}\text{in}\text{the}\text{system}\left(\text{INR}/\text{kg}\right)=25.29/2=12.65$$

Conclusion

In conclusion, vacuum impregnation stands out as a promising technology with the capacity to enhance the nutritional and keeping qualities of fruits and vegetables by incorporating a variety of solutions, such as firming agents, antioxidants, and antimicrobials. To achieve effective vacuum impregnation, the air from the tissue must be extracted as efficiently as possible during vacuum treatment. Thus, the development of an effective vacuum impregnation chamber is of paramount importance. Our developed vacuum impregnation system demonstrated to be effective in maintaining vacuum conditions and infusing spinach leaves with desired solutions substantiated by noticeable enhancement in biochemical parameters. Treated Spinach leaves exhibited color retention and higher antioxidant activity compared to untreated samples.

The adoption of this technology ensure that products with desired parameters can be achieved with minimal impact on their sensory attributes at lower production cost and in a short period. Research needs to be done in the future to optimize the process parameters while taking into account other future combined applications of physical, chemical, and bio preservation technologies, which may allow a better maintenance of the fresh-like qualities of the raw food. Future studies should also take into account consumer acceptance, safety and legal considerations, commercial availability, such as the efficacy, cost-effectiveness ratio, and ease of manipulation of the technology.

Authors’ contributions

AW; conceived this research and designed experiments; wrote the paper and performed experiments and analysis, SKG; conceived, supervised this research, participated in the revisions of it, AK: participated in the design and interpretation of the data, MKT: participated in the design and interpretation of the data, L: participated in the interpretation of the data and revision of manuscript. All authors read and approved the final manuscript.

Funding

Not applicable.

Data availability

Data will be made available on reasonable request.

Code availability

Not applicable.

Declarations

Conflict of interest

We author(s) of the above titled paper hereby declare that the work included in the paper is original and is an outcome of the research carried out by the authors indicated in it. The authors declare that they have no known conflict of interest, financial or personal interests that may have affected this paper’s findings.

Ethics approval

Not applicable.

Consent of participate

All authors have read and approved the MS & the corresponding author herewith consents to review at least 3 manuscripts in my field of expertise.

Consent or publications

Not applicable.