Reducing atmosphere packaging as a novel alternative technique for extending shelf life of fresh cheese

Abstract

An atmosphere composed of hydrogen gas-included gaseous mixture was tested for packaging fresh cheese samples. The cheese samples were packaged in reducing atmosphere packaging (RAP) [RAP 1 (90% CO₂/6% N₂/4% H₂), RAP 2 (50% CO₂/46% N₂/4% H₂)], modified atmosphere packaging (MAP) [MAP 1 (90% CO₂/10% N₂), MAP 2 (50% CO₂/50% N₂) and MAP 3 (Air)], and unpackaged (control) conditions without using any preservatives, then stored at + 4 °C for 7 weeks. The closest values of color and titratable acidity to the fresh sample were observed for RAP 1. The highest and lowest total mesophilic-aerobic bacteria counts were noted for control and RAP 1 samples, respectively. The yeast-mold counts of all sample groups increased by the time; where RAPs groups exhibited the lowest counts. The similarity between RAP and fresh samples attracts attention to the protective role of hydrogen in preserving the freshness of fresh cheese without using any preservatives.

Introduction

White cheese is a popular dairy product in Turkey classified as soft or semi-hard cheese product and ripened in brine. It is generally manufactured from sheep and cow milk, or their combinations (Öner et al. 2006). The shelf life of fresh unpacked cheeses varies according to many factors such as cheese type, storage temperature, and application of heat treatment. Due to its near-neutral pH, high water activity, low salt content, and the presence of oxygen in its environment, the maximum shelf life of fresh cheese was estimated for about 7 days under cold conditions (Del Caro et al. 2012).

Salting and immersion in brine are considered as the main processes for controlling microbial growth, improving characteristic taste and texture properties, and controlling biochemical reactions (glycolysis, proteolysis, and lipolysis, etc.) of fresh cheeses (Guinee 2004). The brine concentration (NaCl, w/w) used in white cheese production in Turkey is about 12–14% (Hayaloglu et al. 2002). Despite its several positive effects, the high sodium chloride intake has been linked to different serious health problems for the consumer such as high blood pressure, chronic kidney and cardiovascular diseases. Since the daily intake of sodium chloride in the industrialized countries is closely linked to the consumption of cheese; the salt content of cheese is advised to be reduced for health considerations (El-Bakry 2012).

Nowadays, there are many preservation techniques used to extend the shelf life of food products; however, packaging has become one of the most preferred techniques thanks to its flexibility and simplicity. The packaging process plays a key role in preventing microbial growth and quality loss and contributing to the sale of packaged products. The consumers demand natural and healthy products, i.e. without chemical preservatives, which guided the food industry to develop a new packaging concept i.e. the modified atmosphere packaging (MAP). The MAP combined with cold conditions has been successfully tested for extending the shelf life of different foodstuffs such as fresh fruits and vegetables, and meat and dairy products (Rodriguez-Aguilera et al. 2011). In this technique; the composition of the air surrounding the packaged food product is actively or passively replaced by a specific gaseous mixture, thus the initial freshness of the food can be kept by reducing the metabolic activity and chemical/biochemical oxidative reactions inside the food item. Carbon dioxide (CO₂), oxygen (O₂) and nitrogen (N₂) are the three main gases used in MAP technique, and the rate of each gas varies depending on the type of food. atmospheric, Noble gases such as argon (Ar) are commercially used for packaging different products such as coffee and snack, but the knowledge on their application and benefits is limited (Pinela et al. 2016; Sandhya 2010). Although vacuum packaging is a good alternative for preserving cheese due to its reduced oxygen content, it negatively affects the texture of product by causing adhesion phenomenon of slices in some cheese varieties such as Mozarella (Alves et al. 1996). On the other hand, MAP was proved to maintain the sensory quality of many different varieties of cheese without damaging the textural structure and inhibit the development of the psychrotrophic bacteria, yeasts, and molds (Eliot et al. 2010).

In the last two decades, several hundreds of studies were performed on the potential use of hydrogen in different topics especially the therapeutic applications. In 2007, a very cited paper (ca. 1000) published in Nature Medicine journal reported the positive role of hydrogen gas (H₂, 4%) as a potential therapeutic antioxidant in preventive and therapeutic applications (Ohsawa 2007). The unique characteristics of hydrogen such as a colorless, odorless, and tasteless, non-toxic, potent antioxidant, gene regulatory abilities, and rapid rates of diffusion across tissue and cellular barriers, as well as its excellent safety record made the hydrogen gas the subject of many recent studies in plant biology, plant tolerance against multiple environmental stresses, disease models, human diseases, and treatment-associated pathologies (Ichihara et al. 2015; Nicolson et al. 2016). Concerning the use of hydrogen gas in food applications, there are few studies covering this topic. Although several different gases have been tested in MAP studies so far, the use of hydrogen gas (H₂) has not been evaluated in the concept of MAP technique. Hydrogen is classified as a food additive with E949, and in EU it is permitted in part C Group I of regulation 1129/2011 additives permitted at quantum satis (Saltmarsh and Saltmarsh 2013). Hydrogen is especially used in margarine manufacturing for its property of saturation of the unsaturated bonds of fatty acids. Moreover, the usefulness of hydrogen application in fruit juice production like orange juice was proved thanks to its protective effect of color and ascorbic acid (Alwazeer et al. 2003). Other reports showed the impact of hydrogen gas on different microbial physiological parameters, decreased oxidative degradation and complete lipid oxidation inhibition of a dairy beverage (Cachon and Alwazeer 2019). The use of H₂-included gaseous mixture in the drying atmosphere for maintaining the sensorial and nutritional quality of apricot and apple was recently reported (Alwazeer 2018; Alwazeer and Örs 2019). Recently two reviews reported the potential applications of reducing atmosphere packaging for extending the shelf life of food products (Alwazeer 2019, 2020).

In the present study and for the first time, the reducing atmosphere packaging (RAP) technique based on the use of gaseous mixtures containing hydrogen gas (H₂) was evaluated for extending the shelf life of the fresh salt-free cheese. The fresh cheese samples were packaged in RAP, MAP and unpackaged (control) conditions without adding salt, brine or any preservatives, and stored at + 4 °C for 7 weeks. The dry matter, fat content, titratable acidity, color (L* and b*) and microbiological (total mesophilic-aerobic bacteria (TMAB) and yeast-mold counts) analyses were evaluated.

Materials and methods

Chemicals and reagents

Amyl alcohol, ethanol, phenolphthalein, calcium chloride, sodium hydroxide, sodium chloride, sulphuric acid, buffered peptone water (BPW), yeast extract agar (YEA) and plate count agar (PCA) were purchased from Merck (Germany), while rennet was provided from a local dairy factory.

White cheese production

White cheese was prepared under laboratory conditions according to Hayaloglu et al. (2002) with slight modifications. Firstly, raw cow’s milk was pasteurized at 72 °C for 15 s followed by a cooling step up to 30–32 °C. The starter culture composed of Lactobacillus delbrueckii subsp. bulgaricus and Streptococcus thermophilus obtained from Ankara University, Food Engineering Department (2%, v/v) as well as calcium chloride (2%, w/w) were added. The inoculated cow’s milk was held for 30 min then liquid rennet (1.5%, v/v) was added. Milk was allowed to stand for 75–90 min to ensure adequate coagulum formation. The formed coagulum was cut then the curd was transferred to a cheese-cloth. A 20 kg weight was applied at room temperature for 24 h to remove the whey. The cheese was then divided into equal parts according to package size.

Packaging process

The slices of fresh white cheese were packaged without adding any brine, salt or chemical preservative. Different gaseous combinations formed of nitrogen (N₂), carbon dioxide (CO₂) and hydrogen (H₂) gases were performed by using a gas mixer (Dansensor, MAP Mix 9001 ME, Denmark). The fresh cheese slices were packaged by a modified atmosphere packaging machine (Lipovak, KV-600, Turkey) using polyethylene laminated polystyrene containers and polyethylene films (Çokay plastik, Turkey) (Fig. 1). Five different gaseous combinations were used for packaging cheese samples as follows: RAP 1 (90% CO₂/6% N₂/4% H₂), RAP 2 (50% CO₂/46% N₂/4% H₂), MAP 1 (90% CO₂/10% N₂), MAP 2 (50% CO₂/50% N₂), and MAP 3 (Air), and a group of samples was left unpackaged (open) under normal atmosphere conditions as a control sample. Both packaged and unpackaged samples were stored at + 4 °C for 7 weeks.

Fig. 1.

Schematic diagram of Reducing Atmosphere Packaging (RAP) process

Total dry matter content

A 5 g of sample was dried in an oven at 105 °C under atmospheric pressure until constant weight, and the results were expressed as a weight (g) in 100 g dry matter.

Total fat content

The total fat content of the samples was performed using the standard Gerber method according to AOAC (2000:18).

Titratable acidity

Firstly, 5 g of the sample was diluted with 20 ml of distilled water then homogenized using a homogenizer (IKA Ultra Turrax, T18, Germany). The mixture was filtered through Whatman No. 4 filter. 2–3 drops of 1% phenolphthalein indicator were added to the 5 ml of filtrate followed by a titration procedure with 0.1 N NaOH until the formation of a permanent light pink color. Results were expressed as lactic acid (%) and calculated using the following equation:

$$\text{Lactic}\text{Acid}\left(\%\right)=\frac{\text{V}\times 0.009}{\text{m}}$$

where V is the amount of 0.1 N NaOH consumed in titration step (ml); m is the amount of sample (g); 0.009 is the milli-equivalent weight of lactic acid (g).

Color characteristics

The color analysis was performed using a colorimeter (Konica Minolta CR 410, Osaka, Japan) where L* is the lightness, while b* represents each yellow (+) and blue (−) in CIELAB system.

Microbiological analysis

10 g of cheese was taken into the stomacher bag, and 90 ml buffered peptone water was added. A homogenization step was applied using a stomacher (BagMixer 400, France). Decimal dilutions were prepared, and 1 ml of appropriate dilution was cultivated on specific media followed by an incubation step. Total mesophilic aerobic bacteria (TMAB) counts were determined on plate count agar after incubation at 37 °C for 48 h, while yeast-molds were performed on yeast extract agar after incubation at 28 °C for 72 h.

Statistical analysis

Statistical analysis was performed using SPSS statistical software (Version 18). One-way and two-way analyses of variance (ANOVA) were applied. Statistical significant differences were considered at the level of p < 0.05. All experiments were performed in four replicates (n = 4).

Results and discussion

Total dry matter and total fat content

The dry matter and fat content of the produced fresh white cheese were found as 42.87 and 13%, respectively. These results agreed with those of Uraz and Şimşek (1998) where they reported a dry matter value of 41.71%. Moreover, Hayaloğlu and Özer (2011) reported a fat content of white cheese ranged from 13 to 28.5%.

Color characteristics

L* value

Table 1 shows that the L* (Lightness) values of cheese samples stored under different conditions (RAPs, MAPs, and control) decreased along the time. The MAP 3 (Air) and control (Unpacked) samples showed the lowest L* values compared to RAP 1, RAP 2, MAP 1 and MAP 2 samples. The presence of oxygen (O₂) in the atmosphere of MAP 3 and control samples causes oxidative reactions changing the color and other physical and chemical properties of the product. The elimination of oxygen and keeping samples away from light exposure were proposed as the main procedures to minimize the damages caused by these reactions. While the difference between MAP 3 (Air) and control samples was significant for the mean values at the end of week 7 (p < 0.05), the decrease in L* value during storage began earlier in the control samples than that of MAP3 (Table 1). The continuous exposition of control samples and the limited exposition of MAP 3 samples to the atmospheric air during the storage period could explain this difference.

Table 1.

L* and b* values of fresh white cheese samples stored at 4 °C under different packaging conditions (RAP, MAP, and control)

Sample group	Time (week)
	0	1	2	3	4	5	6	7	Mean
L* value
RAP1	94.09 ± 0.06aD	93.38 ± 0.59bD	92.37 ± 0.56bCD	93.07 ± 0.60bD	93.54 ± 0.42dD	90.91 ± 0.60cBC	90.31 ± 0.28cAB	88.68 ± 1.2bA	92.04 ± 0.48*c
RAP2	94.09 ± 0.06aF	94.19 ± 0.19bF	93.70 ± 0.23cEF	92.96 ± 0.38bDE	92.28 ± 0.58cdD	90.69 ± 0.43bcC	89.26 ± 0.12cB	86.85 ± 0.1bA	91.49 ± 0.78c
MAP 1	94.09 ± 0.06aD	92.90 ± 0.42bCD	92.93 ± 0.54bcCD	93.04 ± 0.21bCD	91.06 ± 0.23bcC	91.17 ± 1.14cC	89.18 ± 0.72cB	86.66 ± 0.5bA	91.38 ± 0.61c
MAP 2	94.09 ± 0.06aD	92.58 ± 0.29bCD	92.33 ± 0.08bCD	93.29 ± 0.0bD	90.78 ± 1.24bcBC	90.74 ± 0.22bBC	89.83 ± 0.66cAB	87.95 ± 0.9bA	91.45 ± 0.51c
MAP 3	94.09 ± 0.06aE	93.86 ± 0.20bE	93.86 ± 0.12cE	92.42 ± 0.91bE	89.64 ± 0.18bD	87.38 ± 0.38bcC	84.07 ± 0.39bB	68.22 ± 1.6aA	87.94 ± 2.12b
Control	94.09 ± 0.06aE	84.34 ± 0.70aD	75.82 ± 0.22aC	76.38 ± 0.02aC	74.90 ± 0.55aBC	72.89 ± 1.85aB	69.415 ± 0.16aA	69.48 ± 1.4aA	77.16 ± 2.02a
Mean	94.09 ± 0.02*G	91.87 ± 1.04F	90.17 ± 1.95E	90.20 ± 1.87E	88.70 ± 1.91D	87.29 ± 2.0C	85.34 ± 2.24B	80.97 ± 2.6A
b* value
RAP 1	13.86 ± 0.03aA	14.47 ± 0.06aAB	15.01 ± 0.45aBC	15.29 ± 0.95aBC	15.555 ± 0.02aC	15.78 ± 0.57aC	16.77 ± 0.39aD	17.95 ± 0.04aE	15.588 ± 0.32a
RAP 2	13.86 ± 0.03aA	14.8 ± 0.05aB	15.26 ± 0.08aBC	15.82 ± 0.43aC	16.035 ± 0.56bCD	16.95 ± 0.05bDE	17.325 ± 0.75abEF	18.145 ± 0.39abF	16.024 ± 0.35b
MAP 1	13.86 ± 0.03aA	15.34 ± 0.12aB	15.245 ± 0.02aB	16.625 ± 0.12abC	17.585 ± 0.32cdD	18.315 ± 0.1cE	18.33 ± 0.070cE	18.195 ± 0.1abE	16.686 ± 0.41c
MAP 2	13.86 ± 0.03aA	15.27 ± 0.07aB	15.665 ± 0.05aBC	16.3 ± 0.17aCD	16.87 ± 0.040bcDE	18.25 ± 0.64cF	17.78 ± 0.49bcEF	18.685 ± 0.09bF	16.584 ± 0.40c
MAP 3	13.86 ± 0.03aA	16.32 ± 0.06aB	17.34 ± 0.52bC	17.25 ± 0.11bC	18.355 ± 0.13dD	19.435 ± 0.2cE	19.975 ± 0.06dE	20.87 ± 0.28cF	17.926 ± 0.55d
Control	13.86 ± 0.03aA	29.69 ± 0.36bB	32.94 ± 0.22cC	33.18 ± 0.40cC	33.76 ± 0.32eC	35.3 ± 0.49dD	36.615 ± 0.095eE	39.04 ± 0.79dF	31.797 ± 1.88*e
Mean	13.85 ± 0.08A	17.65 ± 1.60B	18.58 ± 1.93C	19.08 ± 1.89D	19.69 ± 1.95E	20.67 ± 2.02F	21.13 ± 2.11G	22.14 ± 2.33*H

Each value is the mean of four replicates (n = 4). Different capital letters (A, B, C, D…) in the same row and different small letters (a, b, c, d…) in the same column indicate a statistically significant difference (p < 0.05). RAP 1: 90% CO₂/6% N₂/4% H₂; RAP 2: 50% CO₂/46% N₂/4% H₂; MAP 1: 90% CO₂/10% N₂; MAP 2: 50% CO₂/50% N₂; MAP 3: Air; control (unpackaged/+ 4 °C); RAP reducing atmosphere packaging, MAP modified atmosphere packaging

*The highest mean value of L* and b* of 7 weeks

The means of L value of the total 7 weeks reveal that each RAP 1, RAP 2, MAP 1 and MAP 2 samples are statistically similar with the highest value attributed to RAP 1 sample (p > 0.05) (Table 1). The similarity of L* value of samples stored under both MAP and RAP conditions reveals the protective effect of the gases used in packaging on preserving the lightness of samples. Temiz et al.(2009) reported that the highest L* value in all packaging combinations belonged to atmospheres consisting of 70% CO₂/30% N₂ and 40% CO₂/60% N₂. Favati et al. (2007) stated that while three independent variables, i.e. gas mixture, storage time and storage temperature, did not significantly affect the color of Provolone cheese, the protective atmosphere stabilized the color during storage.

b* value

Although the b* value increased in all sample groups over the time, at the end of 7th week the lowest and the highest mean b* value were 17.95 and 39.04 for RAP 1 and control samples (Table 1). The means of b * value of different sample groups were as follows: RAP 1 < RAP 2 < MAP 1 = MAP 2 < MAP 3 < control (p < 0.05)(Table 1). The increase in b* value represents the progression in the yellowness indicating that the cheese sample has moved away from its original white color towards the yellow color. The yellow color of the control sample could be clearly detected by the naked eye.

The color of the cheese is linked to two principal components: fat and casein. The color of fat, which is a light yellow, is a combination of three factors: the protein membrane of the beta carotene-rich fat globule, the natural chemistry of the fat‚ and the fat-soluble yellow carotenes that the cow eats (Johnson 1999). Both casein clusters and the protein membranes that surround fat globules in milk hide the yellow beta-carotene color, reflecting white light. Thus, the white color of cheese attributed to the casein clusters appears white. The microbial growth leads to dissolve the protein membrane of fat globule and deform the protein clusters. Moreover, the proteolysis of casein and the pH decrease of cheese due to the microbial growth, as the case of control samples, transform casein to a more soluble form (Johnson 1999). When casein is deformed and no longer reflects white light, the color of the cheese will be dominated by the reflecting components i.e. beta carotene and riboflavin which were originally hidden by the intense reflected white light of casein. This observation was confirmed by the high microbial counts of the cheese control sample and its high TA value (results shown below), while the MAP and RAP samples remained white due to the low microbial growth, the high pH value, and the high moisture content. Furthermore, the dehydration phenomenon that occurred during the storage of control samples in the open-package makes coloring pigments, i.e. beta carotene, more concentrated. MAP3 sample showed also an increase in b* value, i.e. yellowness degree, compared to other MAP and RAP conditions, which was less significant than the control (Table 1).

The protective effect of both CO₂ and H₂ on preserving the whiteness of cheese could be due to the positive protective effect of protein from deformation reactions such as lipolysis. Temiz et al. (2009) reported that the b* value remained unchanged between 17 and 38 days in the Turkish whey cheese (lor) despite the significant effect of storage time and packaging combination. Otherwise, Del Nobile et al. (2009) stated that there were significant differences in color between Ricotta cheese stored under normal and modified atmospheres (50% CO₂/50% N₂; 70% CO₂/30% N₂ and 95% CO₂/5% N₂).

In order to better understand the effect of hydrogen (H₂) gas on the b* value of cheese samples, the RAP 1 samples were compared with MAP 1 (Fig. 2a), and RAP 2 samples were compared with MAP 2 (Fig. 2b). Although both RAP1 and MAP 1, and RAP2 and MAP2 groups contain the same level of CO₂ with 90% and 50%, respectively, RAP 1 showed lower mean b* value than MAP 1, and RAP 2 showed lower mean b* value than MAP 2. This reveals the progression in yellowness (+ b) in white cheese color could be blocked by the presence of reducing gas (H₂) in the gaseous mixture atmosphere.

Fig. 2.

Comparison of means of b* and titratable acidity values between RAP and MAP sample groups of fresh white cheese

On the other hand, although each RAP 1 and RAP 2 samples contain reducing gas (H₂) at the same level (4%), RAP 1 exhibited lower mean b* value than RAP 2 (p < 0.05). This would be due to the high level of CO₂ in RAP1 compared to RAP2 (90 vs. 50%). Otherwise, although the CO₂ level in MAP 1 (90% CO₂) was higher than MAP 2 (50% CO₂), the b* values of both MAP 1 and MAP 2 were similar (p > 0.05). This means that the addition of hydrogen to the gaseous combinations led to a synergistic effect with CO₂, which provided a protective effect of cheese color. On the other hand, the N₂ gas is generally used in packaging technique to replace oxygen in the package atmosphere and to prevent the collapse of the package (Sandhya 2010).

Titratable acidity (TA)

Table 2 shows that the highest and lowest mean values of titratable acidity were found for control and RAP 1 samples, respectively (p < 0.05). Moreover, there was no difference between RAP 2, MAP 1 and MAP 2 samples (p > 0.05). Saldamlı and Kaytanlı (1998) reported a TA value of 0.55% for fresh white cheese, which is close to our results. Hayaloğlu and Özer (2011) reported that the titratable acidity values of white cheese can range from 0.40 to 1.54%.

Table 2.

Titratable acidity values of fresh white cheese samples stored at 4 °C under different packaging conditions (RAP, MAP, and control)

Sample Group	Time (week)
	0	1	2	3	4	5	6	7	Mean
Titratable Acidity (TA %)
RAP 1	0.209 ± 0.002aA	0.219 ± 0.004aA	0.256 ± 0.003aB	0.267 ± 0.003aBC	0.278 ± 0.004aC	0.275 ± 0.002aC	0.302 ± 0.004abD	0.303 ± 0.005aD	0.264 ± 0.008a
RAP 2	0.209 ± 0.002aA	0.245 ± 0.008bB	0.257 ± 0.004aBC	0.263 ± 0.002aC	0.282 ± 0.001aD	0.289 ± 0.005abD	0.305 ± 0.004abcE	0.315 ± 0.00abE	0.271 ± 0.008b
MAP 1	0.209 ± 0.002aA	0.243 ± 0.010bB	0.272 ± 0.006aC	0.275 ± 0.016abCD	0.285 ± 0.001aCD	0.3 ± 0.004bD	0.295 ± 0.002aCD	0.3255 ± 0.002*bE	0.276 ± 0.008b
MAP 2	0.209 ± 0.002aA	0.2405 ± 0.003abB	0.269 ± 0.002aC	0.272 ± 0.002abC	0.288 ± 0.004aD	0.290 ± 0.001abD	0.306 ± 0.002bcE	0.319 ± 0.001bF	0.275 ± 0.008b
MAP 3	0.209 ± 0.002aA	0.2525 ± 0.005bB	0.277 ± 0.004aC	0.301 ± 0.005bD	0.318 ± 0.003bE	0.302 ± 0.008bDE	0.313 ± 0.002cDE	0.341 ± 0.004cF	0.289 ± 0.041c
Control	0.209 ± 0.002aA	0.2875 ± 0.003cB	0.315 ± 0.022bB	0.406 ± 0.012cC	0.494 ± 0.013cD	0.625 ± 0.010cE	0.609 ± 0.001dE	0.618 ± 0.002dE	0.445 ± 0.039*d
Mean	0.209 ± 0.001A	0.248 ± 0.006B	0.274 ± 0.007C	0.297 ± 0.015D	0.324 ± 0.023E	0.347 ± 0.037E	0.355 ± 0.034F	0.370 ± 0.034*G

Each value is the mean of four replicates (n = 4). Different capital letters (A, B, C, D…) in the same row and different small letters (a, b, c, d…) in the same column indicate a statistically significant difference (p < 0.05). RAP 1: 90% CO₂/6% N₂/4% H₂; RAP 2: 50% CO₂/46% N₂/4% H₂; MAP 1: 90% CO₂/10% N₂; MAP 2: 50% CO₂/50% N₂; MAP 3: Air; control (unpackaged/+ 4 °C); RAP: Reducing Atmosphere Packaging; MAP: Modified Atmosphere Packaging

*The highest mean value of titratable acidity values of 7 weeks

In order to verify the effectiveness of the H₂ on the TA, both RAP 1 and MAP 1; and RAP 2 and MAP 2 groups were compared between each other (Fig. 2c, d). RAP 1 samples showed lower mean TA than that of MAP 1. However, there was no significant difference between RAP 2 and MAP 2. According to the evolution of TA values of RAP 1 and RAP 2 samples over each week, no significant difference between them has been detected (p > 0.05). However, in terms of the total mean of 7 weeks, a significant difference was observed between these groups with a lower mean TA value attributed to RAP1 (p < 0.05)(Table 2). Moreover, we note that there was no significant difference between the mean TA values of MAP1 (90% CO₂) and MAP 2 (50% CO₂), which reveals the absence of the effect of CO₂ augmentation on the TA. The significant increase in TA of the control sample could be attributed to the growth of microorganisms in this sample, which are able to produce organic acids as final metabolites.

Microbiological analysis

Total mesophilic-aerobic bacteria (TMAB)

The microflora of cheese can be affected by different factors such as milk quality, the survival of heat-resistant microorganisms during cheese production, and microbial contamination after the process (Del Nobile et al. 2009). According to Table 3, the counts of TMAB increased over the time, and the difference between different package conditions until the 3rd week wasn’t significant (p > 0.05). The lowest and highest mean TMAB counts were observed for RAP 1 and control samples with 1.13 × 10⁶ and 1.38 × 10⁷ CFU/g, respectively (p < 0.05). The high count of TMAB for control samples can be attributed to the development of aerobic microorganisms. Moreover, although MAP 1 contains higher CO₂ than MAP 2 (90% vs. 50%), the two groups showed the same mean TMAB counts (Table 3) (p > 0.05), which reveals the non-significant effect of CO₂ augmentation on TMAB counts when it was alone. However, when the hydrogen gas (4%) was included in the RAP1 and RAP 2 packages, a significant effect was revealed with the lowest mean TMAB counts attributed to the RAP1. This reveals the synergetic effect of H₂ and CO₂ on the TMAB, which rises with the increase of CO₂ level in the gaseous combination. When comparing each MAP1 (90% CO₂) and MAP2 (50% CO₂) samples with control or air (MAP3) ones, we note that these two CO₂-included packages show lower counts of TMAB (p < 0.05). The solubility of carbon dioxide in aqueous phase leads to the formation of carbonic acid and a decrease in the pH value of the product, which causes bacteriostatic and fungistatic effects (Alves et al. 1996).

Table 3.

Total mesophilic-aerobic bacteria (TMAB) and yeast-molds counts of fresh white cheese samples stored at 4 °C under different packaging conditions (RAP, MAP, and control)

Sample Group	Time (week)
	0	1	2	3	4	5	6	7	Mean
TMAB (log CFU/g)
RAP 1	5.57Aab	5.44aA	5.77aAB	5.81aAB	5.91aBC	6.09aC	6.28aD	6.51aE	6.05a
RAP 2	5.57aA	5.46aA	5.89aAB	6.06bAB	6.19bBC	6.37abC	6.59aD	6.89aE	6.35b
MAP 1	5.57aA	5.47aA	5.86aA	6.13bAB	6.27bB	6.65cC	6.97bD	7.04aD	6.57c
MAP 2	5.57aA	5.50aA	5.90aA	6.12bAB	6.27bAB	6.54bcB	6.98bC	7.09aC	6.58c
MAP 3	5.57aA	5.51aA	6.35bA	6.67cAB	7.0cBC	7.20dCD	7.31cDE	7.42bE	7.0d
Control	5.57aA	6.10bA	6.60cAB	6.90dB	7.14dC	7.31eD	7.40dE	7.57cF	7.14*e
Mean	5.57A	5.66A	6.18A	6.45B	6.70C	6.90D	7.07E	7.21*E
Yeast-Molds (log CFU/g)
RAP 1	4.45aA	4.31aA	4.53aA	4.78aB	4.87aB	5.05aC	5.18aD	5.27aE	4.92a
RAP 2	4.45aA	4.33aA	4.76aB	4.91aB	5.09aC	5.18abD	5.29aE	5.49aF	5.08ab
MAP 1	4.45aAB	4.35aA	4.69aB	4.99aC	5.16aD	5.20abD	5.36aE	5.55aF	5.13b
MAP 2	4.45aA	4.35aA	4.81aB	4.96aB	5.11aC	5.27bD	5.33aD	5.51aE	5.12b
MAP 3	4.45aA	4.41aA	5.36aAB	5.49bB	5.61bB	5.83cC	6.14bD	6.21bE	5.77c
Control	4.45aA	5.29bA	6.07bB	6.18cC	6.18cC	6.27dD	6.28cD	6.36cE	6.12*d
Mean	4.45A	4.71A	4.43B	5.55C	5.60C	5.72D	5.83E	5.93*F

Each value is the mean of four replicates (n = 4). Different capital letters (A, B, C, D…) in the same row and different small letters (a, b, c, d…) in the same column indicate a statistically significant difference (p < 0.01). RAP 1: 90% CO₂/6% N₂/4% H₂; RAP 2: 50% CO₂/46% N₂/4% H₂; MAP 1: 90% CO₂/10% N₂; MAP 2: 50% CO₂/50% N₂; MAP 3: Air; control (unpackaged/+ 4 °C); RAP reducing atmosphere packaging, MAP modified atmosphere packaging

*The highest mean value of TMAB and yeast-mold counts of 7 weeks

The low mean TMAB counts found in RAP 1 and RAP 2 samples compared to MAP 1 and MAP 2 (Fig. 3a, b) would be related to the presence of hydrogen (H₂) which decreases the oxidoreduction potential (Eh) of the medium by dissolving in the aqueous phase of the sample. The Eh requirements of microorganisms vary according to their anaerobic and aerobic property; where the anaerobic microorganisms require reduced (negative Eh) conditions to develop, whereas the aerobic microorganisms require oxidized (positive Eh) conditions (Alwazeer 2020). Therefore, positive Eh values allow the growth of both aerobic bacteria and yeast-molds (Arda 2000). Moreover, when the positive Eh values were modified to negative values by applying H₂, the growth of TMAB was inhibited.

Fig. 3.

Comparison of means of total mesophilic aerobic bacteria (TMAB) and yeast-molds counts between MAP and RAP sample groups of fresh white cheese

The CO₂ gas is known to effectively affect the growth of microorganisms and was widely used in packaging of many different food products; however, its high level in MAP1 and MAP2 samples (90% vs. 50%) couldn’t be as effective as the H₂ gas that was used at low concentration (4%). When we compare the mean microbiological results of each of MAP 1 and RAP1 (90% CO₂) samples, and MAP2 and RAP2 (50% CO₂) samples, we note that the effectiveness of H₂ even at low concentrations is potent. This inhibition effect of hydrogen could be linked to its rapid diffusion property into the tissue even in subcellular units such as mitochondria thanks to its very light and small property (MHI 2019b). This feature of hydrogen is utilized in healthcare applications such as inhalation therapy and the treatment of many diseases (MHI 2019a).

Yeasts and molds

Yeasts and molds can contaminate the cheese from various sources such as starter culture, ambient atmosphere, brine, process equipment, and workers. Many varieties of yeasts and molds are associated with the taste, flavor, and appearance of the product; but some species may have a detrimental effect. Yeasts found especially on the surface of cheese can produce different metabolic molecules that adversely affect the aroma, flavor, color, and taste of cheese.

The mean yeast-mold counts were found to be similar for both MAP 1 (90% CO₂) and MAP 2 (50% CO₂) samples (p > 0.05), which reveals the absence of effect of CO₂ augmentation on the growth of these types of microorganisms (Table 3). However, when the two previous combinations were incorporated with hydrogen gas to form RAP 1 and RAP 2, a significant effect was revealed (p < 0.05) (Fig. 3c, d). This demonstrates the synergetic inhibition effect of both CO₂ and H₂ on the growth of yeast-molds. This inhibition effect would also be attributed to the lowering Eh value of the cheese sample phenomenon, which could inhibit the growth of different types of microorganisms.

When comparing each MAP1 (90% CO₂) and MAP2 (50% CO₂) samples with control or air (MAP3) ones, we note that these two CO₂-included packages show lower counts of yeast-molds (p < 0.05). The solubility of carbon dioxide in aqueous phase leads to the formation of carbonic acid and a decrease in the pH value of the product, which causes bacteriostatic and fungistatic effects (Alves et al. 1996). The highest mean yeast-mold counts were observed for the control sample (1.31 × 10⁶ CFU/g) that was in contact with the normal atmospheric conditions i.e. high level of oxygen. In order to keep cheese products away from mold growth, the vacuum or MAP techniques besides the application of chemical preservatives on both packaging materials and product surfaces were proposed (Sørhaug 2011). Kızılırmak Esmer (2009) packaged “Crottin de Chavignol” type goat cheese under both vacuum and modified atmosphere conditions (20% CO₂ + 80% N₂) followed by a storage step at + 4 °C. The author reported that the control samples stored under atmospheric conditions couldn’t be accepted in terms of sensory property at the end of 3rd week due to the growth of visible molds on the surface of cheeses.

Conclusion

The results of this study showed the RAP –packaged white cheese samples were the closest to the fresh samples in the term of quality attributes, thus revealed the effectiveness of RAP technique in preserving the color and microbial property of fresh white cheese without using any brine, salting or preservatives. Although both RAP 1 (90% CO₂) and RAP 2 (50% CO₂) samples contain hydrogen at the same level (4%), the closest values to the fresh sample were observed for RAP 1, which demonstrates the positive protective effect of CO₂, especially at high level, on the preservation of the quality of fresh cheese thus prolonging its shelf life. We remind here that the strategy of CO₂ augmentation was not effective in preserving the quality of white cheese when this gas was used alone i.e. without combination with hydrogen. Otherwise, when this gas was combined with hydrogen (RAPs), the quality attributes of cheese could be protected, thus the shelf life of cheese without using any preservatives could be prolonged. This novel technique of preservation of fresh white cheese can provide a cheese product without salt content, which is important for daily diet, especially, for individuals with sensitivity to high salt concentrations. These high salt concentrations have been reported to adversely affect individuals with high blood pressure, osteoporosis, chronic kidney, and cardiovascular disorders, and it may even increase the risk of developing stomach cancer (HSPH 2019). The results of the present study reveal a potential solution for these individuals by using cheese packaged under gaseous combinations formed from CO₂ and H₂. As the two gases are safe and administrated by the international food administrations to be used in foodstuffs, the consumers can now find their favorable white cheese without health hazards. The encouraging findings of recent research about the potential use of hydrogen gas in therapeutic and preventive purposes confirm the safety but the advantageous use of hydrogen gas in food preservation techniques. There are no safety issues with hydrogen; it has been used for years in gas mixtures in numerous clinical trials without adverse events, and there are no warnings in the literature of its toxicity or long term exposure effects (Nicolson et al. 2016). However, a specific study is advised to be conducted to reveal the effect of the hydrogen-included gaseous combinations on some specific pathogens commonly found in fresh white cheese besides another study concerning the compounds formed during the oxidative reactions of cheese components. The results of these suggested studies should permit researchers to evaluate a sensorial analysis related to RAP technique. It is important to indicate that the infrastructure of the RAP technique is similar to that used by MAP one with no specific machine needs. The materials related to hydrogen gas are only needed. Moreover, the costs of RAP-food products are also similar to that of MAP-products with only a little charge related to hydrogen gas use.