Lyophilized Probiotic Lactic Acid Bacteria Viability in Potato Chips and Its Impact on Oil Oxidation

To produce a new probiotic-containing food product, potato chips, as the most preferred fast food, were chosen. Preferably, it should be preserved for a long period without oxidation. The presented study aimed to compare potato chips containing two lyophilized probiotic lactic acid bacteria (Bifidobacterium longum ATCC 15708 and Lactobacillus helveticus LH-B02) in order to retard lipid oxidation. Lyophilization of probiotics was carried out into two cryoprotective media—skim milk (SM) and gelatin/glycerol (GG) as lactose-free medium. Results revealed that GG and SM media were the most suitable for lyophilization of B. longum and L. helveticus, respectively. The lyophilized live cells were incorporated in potato chips, packed and their effect on oil oxidation was assessed. Results showed that the lyophilized B. longum in SM remained alive at 6.5 log CFU/g for 4 months at 30 °C. Interestingly, potato chip bags containing B. longum lyophilized in SM medium exhibited a decrease in peroxide value (PV) and acid value (AV) of the extracted oil by 40.13% and 25%, respectively, compared to the control bags. The created probiotic potato chips containing B. longum fulfill the criteria of the probiotic product besides the prime quality and sensory attributes.


Introduction
Today, with a busy lifestyle, individuals, especially young and adolescents, prefer to consume 'ready-to-eat' snack foods. The most popular and over-consumed product is potato chips, which are eaten as a snack food, side dish or appetizer. Potato chips may be consumed 3 times/week or more [1,2]. Potato chips are considered a starchy product (total carbohydrate in the range of 60-63.6%) in addition to fat (33-40%) and dietary fibers (1-1.6%) [3][4][5]. Chips also provide other important micronutrients such as sodium (480 mg/100 g) and potassium (166 mg/100 g) [3]. Potato also contains a variety of phytonutrients, most notably carotenoids and phenolic acids, mainly chlorogenic acid [6,7].
By deep oil frying, potato chips absorb a considerable quantity of oil. The final fat content ranges from 35% to 38% based on the total weight [8]. These high oil levels are not only important for nutritional quality but also have a dominant influence on the flavor, calories supplied, and their shelf life [9]. High surface-to-volume ratio of potato chips results in oxidative deterioration of the stored product. Also, lipid oxidation can lead to changes in functional, sensory, and nutritive values that reduce consumer acceptability of the product [4]. As known, the consumption of oxidized oil leads to a variety of diseases, such as atherosclerosis and cancer [10]. The level of oxidation of the containing potato chips oil, and the concomitant formation of off-flavors, is ultimately assessed by some tests such as the peroxide value (PV), acid value (as free fatty acids) (AV) and levels of conjugated dienes (CD) formed during oxidation [11]. Different chemical and natural antioxidants have been examined to retard the lipid oxidation of fats and oils in processed foods [4,12].
Probiotics are live microorganisms that, when administered in adequate amounts, confer health benefits to the host as defined by Food and Agriculture Organization/World Health Organization [13].

Potato Chips Preparation and Frying
Potatoes were washed, peeled, and then sliced into chips (1.2 mm thickness). For more crispness, they were soaked in CaCl 2 (1%) for 10 min. A known amount (c. 2 kg) of refined sunflower oil was placed in a stainless steel pan fryer (50 cm diameter x 20 cm height) and heated at 180 ± 5 • C. The chips were deep oil fried for 5 min, drained to remove excess oil, then cooled [42].

Probiotic Lactic Acid Bacteria
Two probiotic lactic acid bacteria were used in this study. Bifidobactrium longum ATCC 15708 that was obtained from American Culture Collection, Manassas, VA, USA, and Lactobacillus helveticus LH-B02 (Chr. Hansen Laboratory Ireland Ltd., Little Island, Cork, Ireland). Stock cultures were stored at −20 • C in de Man, Rogosa and Sharpe (MRS) broth (Merck, Darmstadt, Germany) containing glycerol (20%). Working cultures were maintained anaerobically in MRS broth at 4 • C and were transferred to a new medium every month.

Probiotic Lactic Acid Bacteria Production
The lactic acid bacteria cells were prepared (before lyophilization) following procedures previously described [43] with minor adaptations. Two cryoprotective media were tested in this study. Skim milk (SM) medium contained 10% skim milk + 5% sucrose while, gelatin/glycerol (GG) medium (lactose free) contained 1.5% gelatin + 1% glycerol (from Sigma Aldrich Co.,Saint Louis, MO, USA). One hundred ml of fresh MRS was inoculated with 2.5 mL of maintained MRS broth culture and incubated anaerobically at 37 • C for 48 h in an Anaerobe Jar. Subsequently, the obtained inoculum was used to inoculate the bottle of MRS (containing 400 mL) and further incubated anaerobically at 37 • C for 24 h (stationary phase). Cells were harvested by centrifugation (Hermle, Z300, Gosheim, Germany) at 11,000× g for 5 min at 4 • C, then washed 3 times with sterilized distilled water. After this, the supernatant was discarded, and the harvested cells were resuspended (final viable counts in the range of 7-8 log CFU/mL) in 100 mL of SM medium. The same procedures were repeated, but the harvested cells were resuspended in 100 mL of GG medium. All the resuspended cells were frozen to −20 • C before the lyophilization.

Lyophilization and Survival Test
The frozen suspensions were dried-frozen (temperature −40 ± 2 • C; vacuum pressure 10 −1 torr for 48 h) using a bench-top lyophilizer (Modulyo bench top freeze dryer, Edwards, Burgess Hill, UK). Viable cell counts were checked after freeze drying and in packed probiotic potato chips at different storage intervals (zero, 1, 2, 3 and 4 months) by the standard plate count method. For this, the dried powder or fried chips was aseptically rehydrated in sterile saline solution (NaCl, 0.85%) at room temperature for 10 min. One ml aliquots were serially diluted and plated; then, MRS agar medium was poured. After incubation for 2-3 days at 37 • C under anaerobic conditions, the colonies were counted and results were expressed as log CFU/g [44].

Probiotic Potato Chips Production and Storage Condition
Potato chips immediately after frying and cooling were pooled together and packaged in 20 × 10 cm polyethylene bags. Each bag was filled with chips (4.5 ± 0.2 g) and lyophilized lactic acid bacteria powder that sticks on the surface of potato chips in the final cell count (10 9 -10 10 CFU/g), vacuumed and heat-sealed then stored at room temperature 30 ± 2 • C in a dark place for 4 months. Bags containing potato chips with no lactic acid bacteria were used as a control. Bags were removed from the storage carton monthly and the lactic acid bacteria viability was assessed as previously described.

Chemical Analysis
Chemical analysis of fresh potato (moisture, protein, fiber, fat and ash) was carried out according to A.O.A.C [45]. Total carbohydrates were determined by the Anthrone method [46] (after HCl hydrolysis, 2.5 N for 3 h), while reducing sugar content was determined by the 3, 5-Dinitrosalicylic acid test (after extraction by hot ethanol solution, 80%) [47].
After the storage period (the fourth month), the stored probiotic chips from 3 bags from each experiment were crushed and weighted. The oil content was determined using the Soxhlet apparatus. Lipid extraction was performed by chloroform/methanol mixture (1:1) as described by Petukhov et al. [42]. Peroxide value (PV) of extracted oil was determined according to Paquot [48] method II.D.13. Briefly, the sample is treated with a mixture of glacial acetic acid and chloroform then with a saturated potassium iodide solution. The liberated iodine is titrated with a standard solution of sodium thiosulfate (0.05 N) and expressed as meq O 2 /kg. Free fatty acid percent (acid value, AV) was determined as described by Atalay and Inanc [49] by titration with KOH (0.05 N) in the presence of phenolphthalein and expressed as Oleic acid. Each analysis was performed in triplicate.

Sensory Evaluation
An untrained panel of students and staff members (n = 20) at Food Science Department, Faculty of Agriculture, Cairo University evaluated the samples monthly for different sensory attributes such as taste, odor, color, appearance, texture and overall acceptance (using a 9-point hedonic scale). Samples were evaluated for their all sensory attributes where 9 and 1 represented liked extremely and disliked extremely, respectively [8].

Statistical Analysis
Experiments were conducted in triplicate and data were analyzed using the CoStat Microsoft program. Significant differences among means (n = 3) were determined by one-way ANOVA, using Duncan's test at p < 0.05.

Survival of Probiotic Lactic Acid Bacteria
Probiotics, being live microorganisms, have a great difficulty being incorporated and still living at the time of food product consumption. Therefore, choosing the right strains, culture conditions, and cryoprotectants should be considered. Protective agents such as skim milk, whey, trehalose, glycerol, sucrose, and glucose are commonly employed to protect bacterial cultures [30]. To preserve lactic acid bacteria during the lyophilization process, two media (i.e., SM and GG) were evaluated as cryoprotectants. The cell viability before and after the lyophilization process are shown in Table 2. It can be observed that the GG medium was better for protecting Bifidobacterium longum after lyophilization, as 79.03% of its initial population was still live. On the other hand, Lactobacillus helveticus lost 66.66% of its viability after lyophilization in SM medium. GG medium had a significant negative effect in the decline of its viability by 92.28%. Lyophilization can cause many negative effects on the cells, such as a disruption of the cell walls resulting from the water vapor transportation to the surface of the sample for sublimation, collapse of protein and shrinkage [31]. Sharma et al. [50] reported skim milk as cryoprotectant for Streptococcus thermophilus strain. However, for Lactobacillus strains lyophilization, Yeo et al. [44] recommended a mix of 10% skim milk and 10% sucrose with 2.5% sodium glutamate. Table 2. Viability of probiotic lactic acid bacteria (LAB) as affected by lyophilization (means ± standard deviation (SD)). Probiotic potato chips as a new probiotic product were assessed. The mean values of lyophilized probiotic cell viability in packed potato chips during storage for 4 months are represented in Table 3. Viability of lyophilized Bifidobacterium longum strain in SM medium gradually decreased (with a significant difference p < 0.05) but still maintained at 6.5 log CFU/g for 120 days. In contrast, in GG medium, departing from 10.14 log CFU/g, it decreased to 7.65 log CFU/g (99.67% loss) after 2 months of storage, then its viability was completely lost. With 10 6 -10 7 CFU/g, potato chips containing B. longum lyophilized in SM medium are considered as a probiotic product because it is the standard limit accepted for probiotic products to be delivered to the consumers at the shelf-life end [16]. In the case of Lactobacillus helveticus strain, in SM medium, the viability decreased reaching 10 4 CFU/g after the storage for four months. On the other side, it could not survive over one month in GG medium. It could be concluded that, under vacuum and high-oil content (potato chips) conditions, skim milk medium could protect both strains for 4 months compared to GG medium. That is supported by the previous results (Table 2), as GG medium caused the highest viability reduction of L. helveticus after the lyophilization step. The presence of milk solids is responsible of the difference of the two cryoprotectants during the storage with potato chips. The high nutrient content of milk might support their growth. Also, lactose, the principal sugar in milk, triggers the production of the inducible enzyme β-galactosidase, which sustains LAB growth [51]. In addition, cryoprotectants rich in amino acids and fermentable sugars (lactose and sucrose) can stabilize the lipid bilayer structure of the cell membrane in the absence of water [16]. Bifidobacterium longum viability in SM was higher than those reported by Nebesny et al. [52], who maintained lyophilized Lactobacillus in chocolate at 5 log CFU/g for 5 months storage at 30 • C. However, Bifidobacterium thermophiles NCIMB 702,554 maintained viability at 7.3 log CFU/g for 90 days at 25 • C [53]. While Guergoletto [25] maintained viable Lactobacillus casei in chocolate bars at 8.3 log CFU/g for 84 days at 25 • C, Mirkovic et al. [54] maintained encapsulated L. plantarum 299v in dark chocolate at 10 8 CFU/g up to 6 months at 20 • C. In another probiotic product, soy protein bars, freeze-dried microcapsules of Lactobacillus acidophilus LA-2 remained in high number throughout 14 weeks at 4 • C [34]. In contrast, in probiotic Lactobacillus plantarum-enriched apple snacks dried by microwave-vacuum, the probiotic bacteria remained above 1 × 10 6 CFU/g for 120 days at 25 • C [35]. Furthermore, freeze-dried immobilized L. casei ATCC 393 cells on casein were detected in yogurt at levels > 7 log CFU/g after cold storage for 28 days [38].

Chemical Evaluation of the Stored Potato Chips
At the end of the storage time, the oil content of the different samples of probiotic potato chips and probiotic-free bags (control) was determined ( Figure 1). The oil content ranged between 23.67% to 24.79%. Remarkably, there was no significant (p > 0.05) difference in the oil content values between probiotic products that contained both strains in all cryoprotective media. The significant difference was observed between B. longum (in SM medium), L. helveticus (in GG medium) with control bags, which is not biologically meaningful (1-1.12%).
days at 25 °C [53]. While Guergoletto [25] maintained viable Lactobacillus casei in chocolate bars at 8.3 log CFU/g for 84 days at 25 °C, Mirkovic et al. [54] maintained encapsulated L. plantarum 299v in dark chocolate at 10 8 CFU/g up to 6 months at 20 °C. In another probiotic product, soy protein bars, freeze-dried microcapsules of Lactobacillus acidophilus LA-2 remained in high number throughout 14 weeks at 4 °C [34]. In contrast, in probiotic Lactobacillus plantarum-enriched apple snacks dried by microwave-vacuum, the probiotic bacteria remained above 1 × 10 6 CFU/g for 120 days at 25 °C [35]. Furthermore, freeze-dried immobilized L. casei ATCC 393 cells on casein were detected in yogurt at levels > 7 log CFU/g after cold storage for 28 days [38].

Chemical Evaluation of the Stored Potato Chips
At the end of the storage time, the oil content of the different samples of probiotic potato chips and probiotic-free bags (control) was determined (Figure 1). The oil content ranged between 23.67% to 24.79%. Remarkably, there was no significant (p > 0.05) difference in the oil content values between probiotic products that contained both strains in all cryoprotective media. The significant difference was observed between B. longum (in SM medium), L. helveticus (in GG medium) with control bags, which is not biologically meaningful (1-1.12%).  The primary products of lipid oxidation are hydroperoxides. Therefore, PV is an importance index to assess the level of lipid oxidation in products containing oils during storage. It reflects the amount of hydro peroxides and secondary oxidation products (ketones and aldehydes) in fat [55]. Peroxide value (PV) of the extracted oil was determined in all samples and represented in Figure 2. Although there were no significant differences in oil content of different probiotic chip samples (Figure 1), the probiotic LAB strain and the lyophilization medium significantly affected the PV values. Interestingly, PV was significantly reduced by 40.13% and 35.47% in bags containing Bifidobacterium longum and Lactobacillus helveticus lyophilized in SM medium, respectively, compared to the control bags (38.85 meq O 2 /kg). In GG medium, lyophilized B. longum strain decreased PV by 19%. This could be explained as this strain remained alive for 4 months in an adequate population. In contrast, the highest PV value was recorded by Lactobacillus helveticus in GG medium (1.71 times higher than the control chips). The explanation of that high increment could be explained as L. helveticus is hydrogen peroxide-producing strain [56]. It may be highly producing H 2 O 2 during storage in GG medium. Hydrogen peroxide is known as an oxidizer [57] and can promote the oxidation of potato chips' oil. On the other side, the antioxidant activity of Bifidobacterium longum to inhibit lipid oxidation was proven by many researchers [58,59]. They explained that activity by the fact that the Bifidobacterium longum strain may secrete an extensive amount of polyphenolic and phenolic compounds that minimized lipid oxidation. In an earlier study, PV ranged from 0.12 to 7.4 meq O 2 /kg fat during two months storage of fried potato crisps at room temperature, which was recorded by Abong et al. [9]. Rababah et al. [12] using grape seed extracts at 1000 ppm minimized the peroxide value development in potato chips during 90 days of storage. The primary products of lipid oxidation are hydroperoxides. Therefore, PV is an importance index to assess the level of lipid oxidation in products containing oils during storage. It reflects the amount of hydro peroxides and secondary oxidation products (ketones and aldehydes) in fat [55]. Peroxide value (PV) of the extracted oil was determined in all samples and represented in Figure 2. Although there were no significant differences in oil content of different probiotic chip samples (Figure 1), the probiotic LAB strain and the lyophilization medium significantly affected the PV values. Interestingly, PV was significantly reduced by 40.13% and 35.47% in bags containing Bifidobacterium longum and Lactobacillus helveticus lyophilized in SM medium, respectively, compared to the control bags (38.85 meq O2/kg). In GG medium, lyophilized B. longum strain decreased PV by 19%. This could be explained as this strain remained alive for 4 months in an adequate population. In contrast, the highest PV value was recorded by Lactobacillus helveticus in GG medium (1.71 times higher than the control chips). The explanation of that high increment could be explained as L. helveticus is hydrogen peroxide-producing strain [56]. It may be highly producing H2O2 during storage in GG medium. Hydrogen peroxide is known as an oxidizer [57] and can promote the oxidation of potato chips' oil. On the other side, the antioxidant activity of Bifidobacterium longum to inhibit lipid oxidation was proven by many researchers [58,59]. They explained that activity by the fact that the Bifidobacterium longum strain may secrete an extensive amount of polyphenolic and phenolic compounds that minimized lipid oxidation. In an earlier study, PV ranged from 0.12 to 7.4 meq O2/kg fat during two months storage of fried potato crisps at room temperature, which was recorded by Abong et al. [9]. Rababah et al. [12] using grape seed extracts at 1000 ppm minimized the peroxide value development in potato chips during 90 days of storage. Acid value (AV) indicates the amount of free fatty acids in the food products and is a sign of oil stability during storage. High AV may cause diarrhea, gastrointestinal discomfort, and even liver damage [60]. In the present study, results in Figure 3 revealed that by lyophilization in SM media, B. longum was the most effective in minimizing free fatty acid formation in potato chips, followed by L. helveticus. Gelatin/glycerol (GG) medium has a negative effect on AV, as chips containing B. longum and L. helveticus formed the most significant FFA percent values (3.22%, 5.98%, respectively) in potato chip bags. Abong et al. [9] reported a significant increase in free fatty acid content in fried Acid value (AV) indicates the amount of free fatty acids in the food products and is a sign of oil stability during storage. High AV may cause diarrhea, gastrointestinal discomfort, and even liver damage [60]. In the present study, results in Figure 3 revealed that by lyophilization in SM media, B. longum was the most effective in minimizing free fatty acid formation in potato chips, followed by L. helveticus. Gelatin/glycerol (GG) medium has a negative effect on AV, as chips containing B. longum and L. helveticus formed the most significant FFA percent values (3.22%, 5.98%, respectively) in potato chip bags. Abong et al. [9] reported a significant increase in free fatty acid content in fried potato crisps during storage at room temperature (25-30 • C) for a total period of 24 weeks. No previous research was conducted to evaluate probiotic lactic acid bacteria in minimizing the peroxide or acid value in potato chips.

Sensory Evaluation of the Stored Potato Chips
Deep frying of potato chips gives them a unique appearance, flavors and texture, leading to a highly palatable product [55]. The mean values of the sensory characteristics of different potato samples are shown in Figure 4. Probiotic potato chips products were found acceptable by the consumers. By increasing the storage time, the sensory evaluation showed significant differences (p < 0.05) between the analyzed samples. Although the packed potato chips containing B. longum and L. helveticus in GG medium had the highest taste and overall acceptability scores at the experiment commencement, it declined to the lowest scores after the fourth month of storage, especially in bags containing L. helveticus in GG medium (overall acceptability score of 4.1). Overall, chips containing B. longum and L. helveticus in SM medium had the highest scores for taste, odor and overall acceptability for the next 3 months compared to chips without LAB (control). The greatest overall acceptability, taste and odor scored by B. longum in SM medium may be correlated to its viability and low PV and AV scores after 4 months of storage. In contrast, the decrease in sensory scores in bags containing B. longum and L. helveticus in GG medium may be related to the off-flavors released during storage. This is probably due to the formed free fatty acids and peroxides in the absence of viable probiotic cells. That means it is technically possible to incorporate B. longum into potato chips bags to preserve its quality during the shelf life.

Sensory Evaluation of the Stored Potato Chips
Deep frying of potato chips gives them a unique appearance, flavors and texture, leading to a highly palatable product [55]. The mean values of the sensory characteristics of different potato samples are shown in Figure 4. Probiotic potato chips products were found acceptable by the consumers. By increasing the storage time, the sensory evaluation showed significant differences (p < 0.05) between the analyzed samples. Although the packed potato chips containing B. longum and L. helveticus in GG medium had the highest taste and overall acceptability scores at the experiment commencement, it declined to the lowest scores after the fourth month of storage, especially in bags containing L. helveticus in GG medium (overall acceptability score of 4.1). Overall, chips containing B. longum and L. helveticus in SM medium had the highest scores for taste, odor and overall acceptability for the next 3 months compared to chips without LAB (control). The greatest overall acceptability, taste and odor scored by B. longum in SM medium may be correlated to its viability and low PV and AV scores after 4 months of storage. In contrast, the decrease in sensory scores in bags containing B. longum and L. helveticus in GG medium may be related to the off-flavors released during storage. This is probably due to the formed free fatty acids and peroxides in the absence of viable probiotic cells. That means it is technically possible to incorporate B. longum into potato chips bags to preserve its quality during the shelf life. Foods 2020, 9, x FOR PEER REVIEW 9 of 13

Sensory Evaluation of the Stored Potato Chips
Deep frying of potato chips gives them a unique appearance, flavors and texture, leading to a highly palatable product [55]. The mean values of the sensory characteristics of different potato samples are shown in Figure 4. Probiotic potato chips products were found acceptable by the consumers. By increasing the storage time, the sensory evaluation showed significant differences (p < 0.05) between the analyzed samples. Although the packed potato chips containing B. longum and L. helveticus in GG medium had the highest taste and overall acceptability scores at the experiment commencement, it declined to the lowest scores after the fourth month of storage, especially in bags containing L. helveticus in GG medium (overall acceptability score of 4.1). Overall, chips containing B. longum and L. helveticus in SM medium had the highest scores for taste, odor and overall acceptability for the next 3 months compared to chips without LAB (control). The greatest overall acceptability, taste and odor scored by B. longum in SM medium may be correlated to its viability and low PV and AV scores after 4 months of storage. In contrast, the decrease in sensory scores in bags containing B. longum and L. helveticus in GG medium may be related to the off-flavors released during storage. This is probably due to the formed free fatty acids and peroxides in the absence of viable probiotic cells. That means it is technically possible to incorporate B. longum into potato chips bags to preserve its quality during the shelf life.

Conclusions
Two cryoprotective media were compared to evaluate their suitability for probiotic lactic acid lyophilization and its protective effect in potato chips. GG and SM media were the most suitable for protecting Bifidobacterium longum and Lactobacillus helveticus, respectively. On the other side, potato chips proved a suitable vehicle for probiotics. Skim milk medium seemed to be the most useful medium in probiotic potato chip production, due to high recorded viability maintenance. Among the tested probiotics, Bifidobacterium longum was the best strain, as it preserved its viability at 10 6 CFU/g for 4 months, and decreased the PV and AV of oil in potato chips. As this strain could limit excessive amounts of reactive radicals in vivo, it may contribute to prevent and control several diseases associated with oxidative stress and over-consumption of potato chips. Potato chips containing B. longum have promising characteristics, with improved stability and sensory characteristics to meet the preferences and demands of consumers. In conclusion, we recommend consuming such invented product in moderation to benefit from the probiotics with their antiobesity influence without impairment to human body.
Ethical Approval: This article does not contain any studies with human participants or animals performed by the author.

Conclusions
Two cryoprotective media were compared to evaluate their suitability for probiotic lactic acid lyophilization and its protective effect in potato chips. GG and SM media were the most suitable for protecting Bifidobacterium longum and Lactobacillus helveticus, respectively. On the other side, potato chips proved a suitable vehicle for probiotics. Skim milk medium seemed to be the most useful medium in probiotic potato chip production, due to high recorded viability maintenance. Among the tested probiotics, Bifidobacterium longum was the best strain, as it preserved its viability at 10 6 CFU/g for 4 months, and decreased the PV and AV of oil in potato chips. As this strain could limit excessive amounts of reactive radicals in vivo, it may contribute to prevent and control several diseases associated with oxidative stress and over-consumption of potato chips. Potato chips containing B. longum have promising characteristics, with improved stability and sensory characteristics to meet the preferences and demands of consumers. In conclusion, we recommend consuming such invented product in moderation to benefit from the probiotics with their antiobesity influence without impairment to human body.
Funding: This research received no external funding.

Conflicts of Interest:
The author declares no conflict of interest.