Effect of Mastic Gum (Pistacia Lentiscus Via Chia) as a Probiotic Cell Encapsulation Carrier for Functional Whey Beverage Production
Food Biotechnology Group, Department of Chemistry, University of Patras, Patras, Greece
*Correspondence to: Terpou Antonia, E-mail: firstname.lastname@example.org
Citation: Terpou A, Bosnea L, Kanellaki M (2017) Effect of Mastic Gum (Pistacia Lentiscus Via Chia) as a Probiotic Cell Encapsulation Carrier for Functional Whey Beverage Production. SCIOL Biomed 2017;1:1-10.
Encapsulation and freeze-drying are considered as the most preferable preservation methods of probiotic cells due to their considerable effects on survival rates. Likewise, the purpose of the present study targeted on the impact of Chios mastic gum (Pistacia lentiscus via chia) as a probiotic cell encapsulation matrix for the production of a novel functional whey beverage. Whey valorization was achieved by the incorporation of different quantities of encapsulated probiotic cells of Lactobacillus casei ATCC 393 in Chios mastic gum. The immobilization bioprocess was performed in whey industrial by-product at 37°~40 ℃ for 24 h. The immobilized biocatalyst was freeze-dried without cryoprotectants. The probiotic cells were successfully encapsulated in the viscus matrix of Chios mastic gum and used for functional whey beverages production. For comparison reasons, whey beverages were prepared with the addition of freeze-dried Chios mastic gum particles at the same quantities as the encapsulated biocatalyst, while initially sweetened whey was used as a control sample. The antimicrobial effect of the incorporated mastic gum can be indicated in all functional beverages due to the reduction of initially detected counts of yeasts and fungi, in comparison to control whey beverage during 30 days of storage at 4 ℃. In contrast to yeast and fungi cell counts that appeared to be decreased, the encapsulated probiotic biocatalyst retained high viability of L. casei (> 1 × 106 CFU/g) during storage. Finally, the organoleptic evaluation revealed the good quality of whey beverages which were also characterized by exceptional masticaroma and flavor. Consequently, the pleasant organoleptic characteristics along with possible probiotic properties of functional whey beverages reveal the dynamics of whey valorization for industrial applications in food production.
Mastic gum, Pistacia lentiscus var. chia, Encapsulation, Probiotics, Functional whey beverages
During the past decade, an increased interest has been noted towards processed foods with incorporated natural food components, targeting to improve consumers well-being . In addition, the past few years' scientists interest has targeted on the production of functional beverages containing probiotics due to many health claims that have been associated with their consumption . Probiotics are microorganisms that when presented as live microbial supplements can confer beneficial affects to the consumer by improving its intestinal balance . In general, there has been established a minimum value of live probiotic bacteria (106 colony forming units per gram of product) to be substantial in products during consumption in order to beneficially modify the gut microbiota of the host . As a result, scientists have targeted on the development of different methods, like cell immobilisation, microencapsulation or drying (freeze-drying, spray-drying), in order to reinsure or enhance probiotic cell viability during manufacture and product storage . Finally, the combination of drying and encapsulation there can be achieved excellent properties of probiotic cell protection, stabilization, solubility and even controlled release of the encapsulated cells .
Mastic gum is a natural resin derived from the stem and the leaves of the mastic tree, Pistacia lentiscus Linn, native to Mediterranean areas. The beneficial, healing properties of mastic have been known since antiquity. For example, mastic gum has been used in traditional Greek medicine for various gastrointestinal disorders like dyspepsia or peptic ulcer . Likewise, current research suggests that Chios mastic gum (Pistacia lentiscus var. chia) possesses many beneficial (antimicrobial, antioxidant, hepatoprotective) properties . More specifically, it has recently been noted that some specific components of mastic gum like terpenoids are associated with the resins' anti-inflammatory and antimicrobial effects while natural phenols and flavonoids are related to potential antioxidant activities . In addition, Chios mastic gum was traditionally regarded as an anti-cancer agent  while these old beliefs have recently been confirmed by scientific evidence showing that mastic can induce apoptosis and possess antiproliferative activity in colon cancer cells . Chios mastic gum, in addition to its therapeutic effects, is also commonly used in the food industry as a food additive, herbal remedy or most commonly as chewing gum . More specifically, mastic gum has been extensively used as a chewing gum base, as a flavoring additive in ice-cream, as a texture modifier in baked products, sparkle water additive and for confectionery and liqueurs [11-13]. In addition, recent studies have demonstrated the use of mastic gum as a microencapsulating matrix forming material for sustained drug release .
Cheese whey is the main liquid by-product of the dairy industries remaining after milk coagulation and removal of the curd during cheese manufacture. Cheese whey is usually discarded as a waste to the environment, resulting in major polluting problems due to its high organic load . According to FAO (Food and Agricultural Organization) cheese is one of the main agricultural products worldwide. It has been estimated that due to continuously increasing cheese production at an approximate rate of 2% each year, cheese whey quantities increase as well (FAO, 1999) . Apart from the high organic load which is mainly responsible for environmental pollution, cheese whey can be suitable for further treatment since it contains about 8% solids (50% of total solids of the initial milk) including about 20% of the protein, lactose, minerals, water-soluble vitamins, lactic acid and trace elements. As a result, whey valorization has been constantly increasing the past few years targeting on the production of added-value products, such as ethanol , lactic acid , electricity , whey powder, nutritional whey proteins, bioactive peptides , single cell protein , biodiesel , probiotic biocatalysts , dairy starter cultures , dairy beverages  etc.
According to the present study whey valorization could be achieved by Chios mastic gum fortification targeting to improve the nutritional quality of novel whey beverages, characterizing it as a new age food product with plethora of probable health benefits. According to Triantafyllou, et al.  the therapeutic dose of Pistacia lentiscus mastic ranges between 1~5 g/person/day while even a quantity dose of 28 g/person/day (0.67% of a daily diet) could not be not toxic for the consumer . Nevertheless, in the present study the quantities of mastic gum used for whey fortification retained up to 1.2 g/100 mL of beverage. These recent findings support the concept of combining a probiotic strain like L. casei ATCC393 with an encapsulation support like Chios mastic gum which can be used in parallel as a natural biopreservative for functional beverage production. The beneficial synergies between mastic gum antimicrobial properties and bioactive components like probiotic bacteria and their incorporation in whey have the potential of a new era in industrial by-product valorization targeting functional food innovations [2,28,29].
Materials and Methods
Probiotic cells encapsulation bioprocess
The probiotic, Gram+, Lactobacillus bacterial strain Lactobacillus casei ATCC 393 (DSMZ, Braunschweig, Germany) was cultivated and used for the encapsulation bioprocess. The probiotic bacterial strain was selected according to the in vivo and in vitro studies reinsuring its survival in gastrointestinal tract along with adhesion to the intestine and modulation of intestinal microflora in rats .
Wet biomass was harvested by centrifugation (Sigma 3K12, Bioblock Scientific, France) at 5000 rpm for 10 min. For preparation of the encapsulated biocatalyst the probiotic wet biomass was introduced in sterile whey and fermented at 37 ℃ for 24-48 h along with freeze-dried particles of mastic gum . The fermented liquid was decanted and the encapsulated biocatalyst was washed twice with sterile Ringer's ¼ solution for the removal of free cells.
Verification of probiotic cells encapsulation by scanning electron microscopy and microbiological analysis
Samples of the encapsulated biocatalyst were examined by Scanning Electron Microscopy (SEM) in order to reinsure immobilization. For comparison reasons, samples of mastic gum were also observed. The samples of the encapsulated biocatalyst were freeze-dried at 5 × 10-3 bar and at -45 ℃ in a Freeze-Drying System, Freezone 4.5 (Labconco, Kansas City, Missouri, USA) until total moisture removal (48-72 h). All dried samples were coated with gold in a Balzers SCD 004 Sputter coater (Bal-Tec, Schalksmuhle, Germany) for 2 min and examined in a JSM-6300 scanning electron microscope (JEOL, Tokyo, Japan).
For the enumeration of the encapsulated L. casei cells, 10 g of the biocatalyst were blended with 90 mL of sterile Ringer's solution (1/4 strength). The suspension was serially diluted (ten-fold), plated on MRS agar and incubated at 37 ℃ for 72 h .
Functional whey beverage manufacture
Cheese whey (5% lactose) was obtained by a local cheese factory (A.VI.GAL SA-Achaia milk industry) as an industrial by-product of Feta cheese manufacture, after protein removal. The whey was filtrated and pasteurized at 65 ℃ for 30 min and then temperature was left to drop at 37 ℃. The pH value was adjusted to 3.9 by the addition of 10% citric acid . Citric acid is a food grade material used as an aromatic and preservative in foods and non-alcoholic beverages [32,33]. Whey was enforced by 15% of glucose syrup (85 ℃ for 15 min). The sweet whey beverage was placed into 5 equivalent sterile glass containers and allowed to cool.
The first sample was used as a Control Whey Beverage (CB) containing only deproteinized whey and the sweetener. The second and third sample were enforced by different amounts of the encapsulated biocatalyst, while the forth and the fifth sample were enforced by different amounts of mastic gum. The encapsulated biocatalyst and the particles of mastic gum were freeze-dried prior use at 5 × 10-3 bar and at -45 ℃ in a Freeze-Drying System, Freezone 4.5 (Labconco, Kansas City, Missouri, USA). Freeze drying was applied at both the immobilized biocatalyst and mastic gum particles in order for mastic to be pulverized and easily incorporated in cheese whey . The second sample was prepared by the addition of the encapsulated biocatalyst incorporated at a concentration of 0.5 g per 100 mL of whey beverage with continuous stirring (IB1). Respectively, the third sample was prepared by the addition of 1.2 g per 100 mL of whey beverage (IB2). For comparison reasons, whey beverages were prepared only with the addition of freeze-dried mastic gum of 0.5 (MB1) & 1.2 (MB2) g per 100 mL of whey beverage respectively. All beverages were stored at 4 ℃ for 30 days.
Whey beverages microbiological profile and probiotic cell viability
On each test day 10 mL portion of each whey beverage was diluted into 100 mL of sterilized Ringer solution ¼ strength and mixed in a stomacher (Bagmixer 400, Model VW, Interscience). Subsequently, the appropriate serial dilutions were prepared by sterile Ringer's solution ¼ strength (LabM, United Kingdom). Viable cell counts of Lactobacillus casei ATCC393, yeast & fungi, enterobacteria and staphylococci were enumerated on the selective media of each species respectively: (i) MRS agar, 37 ℃, 48 h, (ii) Potato Dextrose Agar, 30 ℃, 48 h, (iii) Violet Red Bile Glucose Agar-VRBGA, 37 ℃, 24 h and (iv) Baird Parker agar, 37 ℃, 48 h according to instructions given by the manufacturer (LabM, United Kingdom).
All samples were homogenized prior to analysis. The pH of all whey beverages was measured using a digital pH-meter by direct immersion of the electrode (ΕPI-BION SENTRON pH-System 1001). Titratable acidity was determined by titration using 0.1 mol L-1 NaOH and phenolphthalein as an indicator and expressed as g of lactic acid per 100 g of whey beverage.
Alcohol (ethanol) and residual sugars (glucose, glactose) were determined by high performance liquid chromatography, using a Shimadzu HPLC chromatograph consisting of an SCR-101N stainless steel column, an LC-9A pump, a CTO-10A oven at 60 ℃, and an RID-6A refractive index detector. Three times distilled water was used as mobile phase with a flow rate of 0.8 ml/min and butanol-1 was used as an internal standard at a concentration of 0.05% v/v. Quantities of 40 μl of the samples diluted to 1% v/v, were injected, after filtration, directly into the column. The alcohol and residual sugar concentrations were calculated using standard curves.
Whey beverages after the 1st day of production were placed into glass containers of equivalent amounts (100 mL), coded randomly by three-digital numbers and served cold (4~6 ℃). Sensory evaluation was carried out by 10 laboratory members, priory trained, using locally approved protocols. Evaluators used water to clean their mouth between sample testing. The panel was asked to evaluate each beverage on a 0-10 scale (the higher the number the greater the intensity) based on cheese whey odor, mastic odor, smoothness, sweetness, sour taste, bitterness, color, and overall acceptability. Results are presented as a star chart of the product's attributes.
Experimental design and statistical analysis
Whey beverage manufacture and analysis was carried out in triplicate and results are presented as mean values ± standard deviation. All experiments were designed and analyzed statistically by ANOVA. The significant differences among results (coefficients, ANOVA tables and significance) were computed using SPSS v.8.5.
Results and Discussion
The novel probiotic encapsulated biocatalyst
Electron micrographs reinsured encapsulation of the probiotic cells within the matrix of Chios mastic gum (Figure 1). The average encapsulation yield was reported by microbiological analysis of the encapsulated biocatalyst presenting an average of 1.5 g of L. casei ATCC393 cells successfully encapsulated per 100 g of mastic gum (data not shown). Subsequently, 4 log CFU of L. casei cells were proved to be encapsulated in each gram of Chios' mastic gum.
Figure 1: Electron micrograph (SEM) presenting Lactobacillus casei ATCC393 cells entrapped within the viscus matrix of Chios mastic gum (Pistacia lentiscus via chia). View Figure 1
Microbiological characteristics of functional whey beverages
All whey beverages were tested for staphylococci and enterobacteria cell counts and the results showed no detection of such pathogenic microorganisms (data not shown) throughout the hole storage period (30 days, 4 ℃). On the other hand, it should be noted that there were detected viable cell counts of yeast and fungi in all whey beverages at the 1st storage day at 4 ℃. At this point it should be illustrated that in all functional whey beverages with incorporated particles of Chios mastic gum either free or with encapsulate probiotic cells the counts of yeast and fungi were eliminated by the end of storage period (Figure 2). Yeast and fungi are characterized by many studies as possible spoilage microorganisms in many dairy products . So, it's of high possibility that antimicrobial agents of mastic gum  in addition with the antagonistic effect of L. casei cell counts could have eliminated the initial population of yeast and fungi for the novel functional beverages compared to control beverage samples. According to  it has been initiated that a majority of volatile substances, such as essential oils and plant extracts can increase food microbial stability if they are applied in the headspace of the packaging. So, we can conclude that natural volatile antimicrobial compounds through various carriers can motivate the food industry into adopting such innovative technologies avoiding in parallel the risk of chemical preservatives that can act as possible carcinogenic agents.
Figure 2: Counts of yeast & fungi in whey beverages during 30 days of storage at 4 ℃. Half bars on columns represent the standard deviation of the means of triplicate experiments; Different superscript letters on columns for the same treatment indicate significant differences (P < 0.05) among the samples. View Figure 2
According to the microbiological analyzes of probiotic cell counts (Table 1) carried out during storage for 30 days at 4 ℃, L. casei was found to retain its viable cell counts at a concentration over 106 log CFU/mL. In Table 2 is presented the viability of the probiotic cells that were incorporated in whey beverage by the addition of the encapsulated biocatalyst in different amounts. It was noted that in the case of incorporation of 1.2 g of the encapsulated biocatalyst the viability of probiotic cell was higher compared to the samples with incorporated 0.5 g of the encapsulated biocatalyst. These results characterized all novel functional whey beverages with the encapsulated biocatalyst as probiotics [37,38]. It is noteworthy that the encapsulated probiotic cells did not seem to be affected by the presence of mastic gum retaining in high population during the hole storage period. Additionally, we can assume that encapsulation acts protectively to probiotic cells against storage conditions and whey beverage acidic environment since both whey beverage with the encapsulated biocatalyst showed higher survival rates during storage. These results indicate the extended shelf-life of functional whey beverages prepared by encapsulated probiotic cells due to the absence of enterobacteria, staphylococci, yeast and fungi by the end of storage period. In all cases in vivo trails should be performed in the future in order to reinsure probiotic effects of the novel whey beverages.
Table 2: PH, total acidity, glucose, galactose and ethanol content in whey beverages during 30 storage days at 4 ℃. View Table 2
Whey beverage physicochemical characteristics
The physicochemical characteristics of whey beverages are presented in Table 2. In all cases deproteinated whey used as the raw material for the production of functional whey beverages. The initial pH value was of 6.43, lactic acid 1.02 g/100 mL whey, lactose 4.83 g/100 mL, glucose 0.04 g/100 mL and galactose 0.02 g/100 mL. The pH of the initial cheese whey was adjusted to 3.90 by the addition of citric acid solution. It is noted that no important difference was observed concerning the acidity of whey beverages during 30 days of storage at 4 ℃. In general, whey beverage's physicochemical characteristics ranged within acceptable levels during 30 days of storage .
Another fact that might be noteworthy is that there was observed a small increase of ethanol production in the control whey beverage by the end of storage period most likely as a result of yeast and fungi viable cell counts. In parallel to ethanol production, was observed in control samples, reduction in glucose content mostly likely as a result of yeast and fungi accumulation. Finally, galactose content ranged in usual levels of whey beverages while no significant differences were observed within the samples.
Sensory evaluation of functional beverages
Whey beverages were tested for their organoleptic characteristics and the results are presented in Figure 3. Evaluation was performed on 50 mL whey beverages on the first day of preparation after cooling all samples at 4 ℃.
It appears that the use of Chios mastic gum in the preparation of whey beverages significantly affected (P < 0.05) the preferences of the tasters, without their preferences being significantly influenced by the presence or absence of L. casei cells. At this point it is important to mention that all tasters could detect the taste and aroma of mastic gum in whey beverages but could not feel any difference in smoothness compared to control beverages. This came as a result of mastic gums' freeze-drying that resulted in pulverizing the resins microparticles and were not detectible when incorporated in whey functional beverages. In fact, all whey beverages with incorporated Chios mastic gum (free or with encapsulated probiotic cells) were characterized as cool beverages with a pleasant characteristic aroma. The industry should focus on increasing the flavor acceptance of these products, as the flavor is directly related to the overall acceptance and should also develop the attributes that were related to the most accepted samples. This result brings practical information to the industry, indicating which attributes are related to a possible acceptance or rejection of products. Thus, the sensory characterization of products supports the area of research and development that is directly linked to product sales, all aiming at a more accepted product, increasing company's sales.
The frequency of consumption of a product is directly linked to its characteristic attributes accepted by most consumers, and the overall acceptance of the product. Therefore, to increase the consumption of a product the industry should invest in its development to adapt its sensory characteristics to the population habits as well as publicize the product, generating an increase in the company's revenue.
The possibility of developing a novel probiotic and antimicrobial beverage by industrial whey valorization, is most possible. Regarding the freeze-dried biocatalyst, is reinsured that the probiotic cells were successfully encapsulated in the viscus matric of Chios mastic gum retaining their viability despite the resins' possible antimicrobial properties. The obtained results showed that the encapsulation bioprocess favored the viability of L. casei using cheese whey as a fermentation media while in parallel L. casei viability maintained in high counts during refrigerated storage. In addition, the possible antimicrobial properties of Chios mastic gum with the synergistic effect of L. casei cells resulted in shelf-life extension of whey beverages by minimizing yeast and fungi viability. Consequently, the pleasant organoleptic characteristics along with the probiotic properties of these novel whey beverages reveal one of the many dynamics of whey valorization for functional food production.
Dr. Antonia Terpou would like to thank IKY fellowship of excellence for postgraduate studies in Greece - SIEMENS PROGRAM.
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