Helicobacter pylori in ice cream and its control using mastic gum essential oil

Objective: This study aimed at assessing the prevalence of Helicobacter spp. in ice cream sold at Qena city (Egypt) with special reference to H. pylori, and to study the antimicrobial effect of mastic gum essential oil on H. pylori. Materials and methods: A total of 60 small and large scale ice cream samples (30 each) were investigated for the presence of Helicobacter spp. Then, the essential oil of mastic gum was added to lab prepared ice cream inoculated with H. pylori isolate in a count of Log6 using 2 concentrations of 0.8 and 1.6% and one group was kept as a control group. Each group was subdivided into 2 sub-groups; one was kept at -5°C and another one was kept at -20°C. Results: Helicobacter spp. could be isolated from 11.7%, while, H. pylori could be found in 3.3% of the total examined ice cream samples. Regarding the antiHelicobacter pylori effect of mastic gum essential oil added to lab prepared ice cream, in samples kept at -5°C it was found that after hardening the count fall into Log5 in both control and 0.8% groups and log4 in 1.6% essential mastic oil concentration. Reduction in H. pylori at the first week and third day of storage reached 100% where H. pylori couldn’t be counted or isolated from both 0.8 and 1.6% concentrations, respectively. While in ice cream samples stored at -20°C, the reduction in H. pylori load was rapid at first, it was log4 and log3 in groups of 0.8% and 1.6% concentrations, respectively versus log5 in control samples. At the third day H. pylori was not able to be counted or isolated at third day of storage for both concentrations. Conclusion: All isolates recovered from small scale ice cream samples reflexing the hygienic conditions under which samples were produced. Mastic gum essential oil exhibited a powerful anti-H. pylori effect recommending its addition to food matrix for therapeutic purposes or as a functional food.


INTRODUCTION
Helicobacter pylori is considered as the causative agent of peptic ulcer, chronic gastritis, gastric adenocarcinoma, mucosa associated lymphoid tissue lymphoma and duodenal ulcer (Sachdeva et al., 2014), and it is the first recognized carcinogenic bacteria causing gastric cancer (Dore et al., 2001;Rahimi and Kheirabadi, 2012;Momtaz et al., 2014;Mousavi et al., 2014;Yahaghi, et al., 2014).H. pylori is thought to be the second dominant cause of cancer worldwide (WHO, 2010).Existence of H. pylori in the stomach of domestic animals, milk, meat, and gastric biopsies indicates that domestic animals and the food originated from them may act as potential sources of this bacterium (Mousavi et al., 2014).Composite foodstuffs, particularly milk, have been considered as a prospective source of human infection (Fujimura et al., 2002) due to its acidic pH, nutritional values, salt concentration and presence of high amount of activated water that assist the growth and survival of H. pylori for long time, and subsequently transmit to human (Fan et al., 1998;Quaglia et al., 2007).The ways by which H. pylori is transmitted have not been definitely assured, however, some studies indicated that oral-oral or feco-oral way of transmission may occur (Brown, 2000;Calvet et al., 2013).Food may serve as a vehicle for H. pylori transmission through primary contamination of food originating from animal or secondary contamination due to improper handling by human (Quaglia et al., 2007)..For the effective treatment for H. pylori infections, two antibiotics and a proton pump inhibitor are traditionally used.However, the use of these antibiotics for long time may result in the development of resistance towards H. pylori, and also may change the normal flora of the gastrointestinal system (Murali et al., 2014), and subsequently may limit in the therapeutic options (Mégraud, 2004).Since consumers nowadays are more selective for their food items and are more demanding for natural antimicrobials, researchers are deeply involved in searching of natural antimicrobial alternatives.One of the most famous natural antimicrobials is plants' essential oil particularly essential oil of mastic gum which is Generally Recognized as Safe (GRAS) (Lucera et al., 2012).
Mastic gum is the resin of Pistacia lentiscus, and has been used to treat abdominal pain, dyspepsia and gastric and duodenal ulcer (Al Said et al., 1986).The essential oil of mastic gum is a valuable product named mastic oil comprising of a mixture of a several compounds, and most of them have antimicrobial properties (Tassou and Nychas, 1995;Magiatis et al., 1999).It is excessively used in confectionery products as food additive, in the perfume manufacture and also as a component in cosmetics and health products.Mastic oil displays a variety of pharmacological properties, such as antimicrobial (Koutsoudaki et al., 2005;Paraschos et al., 2011), anti-inflammatory (Heo et al., 2006), and antileukaemic (Loutrari et al., 2006).
The anti-H.pylori activity in vitro of mastic oil has been documented by several researchers (Koutsoudaki et al., 2005;Paraschos et al., 2011;Miyamoto et al., 2014).Since food has complex media and the interactions between antimicrobials and food macromolecules, particularly proteins and fat, may influence the performance of the antimicrobial activity (Shelef, 1983), the antimicrobial effect of mastic oil on H. pylori inoculated in ice cream was investigated in this study.To our knowledge, this is the first report concerning the anti-H.pylori effect of mastic essential oil in dairy food matrix.The present work aimed at isolating and confirmation of H. pylori from ice cream samples sold at Qena (Egypt) markets, and to investigate the anti-H.pylori activity of mastic gum essential oil on H. pylori inoculated in lab prepared ice cream.

MATERIALS AND METHODS
Samples: A total of 60 small and large scaled ice cream samples (30 each) were examined for the presence of H. pylori.The samples were collected from different localities at Qena city, Egypt, and were transferred in ice box to the lab as soon as possible for further studies.

Isolation of H. pylori from ice cream: Isolation of H.
pylori was done according to the technique described by Stevenson et al. (2000).Melted ice cream samples were enriched using H. pylori Special Peptone Broth (HPSPB) supplemented with 5% sterile horse serum and H. pylori selective supplement (Oxoid, SR0147E) (5 mg/L vancomycin, 2.5 mg/L trimethoprim, 2.5 mg/L cefsulodine and 2.5 mg/L amphotericine B).Incubation was done at 37°C for 48 h in an atmosphere of 6% O2, 10% CO2 and 84% N2, which supplied by gas generating kits (Oxoid BR 56) in an anerobic jar.Then, HPSPA supplemented with 5% sterile horse serum and SR0147E was streaked using a loop full of the enrichment broth of each sample.The plates were incubated at 37°C for 3-7 days under the same microaerobic condition as mentioned before.All the cultured plates were inspected after 3, 5 and 7 days.Suspected colonies grew slowly and recognized by its small shape not exceeding 2 mm in diameter, circular, convex and translucent as described by Naclmmkin and Skirrow (1998).The obtained isolates were identified morphologically and biochemically as clarified by Zenner (1999).The bacterial culture showing urease positive reaction and negative reaction of hippurate hydrolysis and nitrate reduction was identified as H. pylori.

Confirmation of H. pylori isolates by PCR:
QIAamp DNA Mini kit (Qiagen, Germany, GmbH) was used to preform DNA extraction from H. pylori according to the manufacturer's instructions with slight modification.In brief, 200 µL of the sample suspension was incubated with 10 µL of proteinase K and 200 µL of lysis buffer at 56°C for 10 min.Next, 200 µL of 100% ethanol was added to the lysate and samples were washed and then, centrifugation was done.Finally, nucleic acid was eluted with 100 µL of elution buffer.PCR was done to confirm the H. pylori isolates using the primer sequences (EHC-U; 5'-CCCTCACGCCATCAGTCCCAAAAA-3' and EHC-L; 5'-AAGAAGTCAAAAACGCCCCAAAAC-3'), as described by Van Zwet et al. (1993).The primers were synthesized from Metabion (Germany), and were used for the amplification a 417-bp fragment of the flanking region of H. pylori ureC gene.The PCR mixture reaction (25 µL) consisted of 12.5 µL of Emerald Amp Max PCR Master Mix (Takara, Japan), 2 µL of each primer of 20 pmol, 6.5 µL of water, and 2 µL of DNA template.Amplification of DNA was accomplished with 30 cycles of the following: primary denaturation was at 94°C for 10 min, annealing at 62°C for 1 min and extension at 72°C for 1 min, with a final extension at 72°C for 10 min.The PCR products was separated by electrophoresis on 1.5% agarose gel (Applichem, Germany, GmbH) in 1xTBE buffer at room temperature and gradients of 5V/cm were used.Gel analysis was accomplished by loading 30 µL of the products in each gel slot and a 100-bp DNA Ladder (Qiagen, Germany, GmbH) was used to determine the fragment sizes.Finally, photographing of the gel was carried out by a gel documentation system (Alpha Innotech, Biometra).

Extraction of mastic essential oil:
The essential oil was extracted from the raw samples by steam distillation of mastic gums and water mixture in ratio of 1:10 for 5 h at 1.5 pressure and 105-110°C (Miyamoto et al., 2014).Part of the mastic gum used in this study was obtained as a gift and the other part was bought from different herbal markets.

Detection of anti-H. pylori effect of mastic gum
essential oil: Fresh culture of H. pylori was adjusted with sterile distilled water to obtain turbidity equivalent to 0.5 McFarland turbidity standards (1.5x10 8 /mL).One millilitre of H. pylori culture suspension was used to inoculate Muller Hinton agar supplemented with 7% horse blood without antibiotics obtaining a count of 1.5x10 7 cells/mL.Then blank paper disks were placed on the surface of the inoculated agar plates and saturated with 20 μL of mastic oil and incubation at 37°C for 48 h in an atmosphere of 6% O2, 10% CO2 and 84% N2, using an anerobic jar and gas generating kits (Oxoid BR 56).
The antibacterial activities were estimated by measuring a diameter of the inhibition ring (Miyamoto et al., 2014)

Detection of MIC and MBC of mastic gum essential oil:
The inhibitory effect of mastic gum essential oil on H. pylori was examined using Broth Dilution Method, as described by Gkogka et al. (2013).In brief, sloppy agar was made by preparing a Muller Hinton broth supplemented with 7% horse blood without antibiotics and with the addition of 0.2 % w/v bacteriological agar.Serial two-fold dilutions of mastic essential oil (0.1, 0.2, 0.4.0.8, 1.6, 3.2, 6.4 and 12.8% v/v) were prepared by vortexing the different concentrations of essential oil with the sloppy agar.Then, 1 mL of the bacterial suspension was added in each sloppy agar tube (containing 1 mL sloppy agar) to obtain a final concentration of log7/mL, and the used concentrations of mastic oil mentioned above were used in duplicate for achieving final concentrations in the range of 0.05 to 6.4% v/v. 2 negative controls were prepared (one is containing mastic oil without bacterial inoculation and another one is inoculated with H. pylori without addition of mastic gum oil).After incubation under the condition described above, the Minimal Inhibitory Concentration (MIC) that is the lowest concentration of mastic oil that results in no growth of the inoculum and the Minimum Bactericidal (MBC) is the minimum concentration that kill bacteria was determined by plating the clear tube and the first concentration that showed turbidity.

Manufacturing of ice cream in the laboratory:
Ice cream was prepared using ice cream powder and cool sterilized milk according to label instruction of the producer.Then the prepared ice cream was divided into 3 groups, each group consisted of 2 portions (100 mL each).Inoculum of H. pylori was added to each portion of the 3 groups to obtain count of log6 CFU/mL after through mixing.Then, a group was treated with 0.8% v/v of mastic oil, while another group was treated with 1.6% v/v.The last group was kept as control.One portion from each group kept at -5°C and the other one kept under -20°C.Samples were taken to detect the count of H. pylori after hardening and for the first 3 days consequently, and then every week for 4 weeks.H. pylori was enumerated by surface plating method, using Muller Hinton agar supplemented with 7% horse blood and SR0147E.The inoculated plates were incubated at 37°C for 48 h in an atmosphere of 6% O2, 10% CO2 and 84% N2, using an anerobic jar and gas generating kits (Oxoid BR 56).

RESULTS AND DISCUSSION
According to data showed in Table 1, Helicobacter spp.could be isolated from 23.3% of the examined small scale ice cream samples.While none of the ice cream samples belonged to large scale was harbouring Helicobacter spp.Helicobacter spp.could be isolated from 11.7% of the total examined ice cream samples.Incidence of Helicobacter spp.obtained by El-Khawaga (2006) was higher, also, Mokhtar (2004) found higher percent (30%) of Helicobacter spp. in the examined ice cream samples.
Helicobacter spp.were biochemically identified as H. pylori, H. pullorum and H. cinaedi with an incidence of 6.7, 13.3 and 3.3% of the examined small scale ice cream samples and 3.3, 6.7 and 1.6% of the total investigated ice cream samples, repectively (Table 1).Regarding incidence of H. pylori, it was found that El-Khawaga (2006) reported similar incidence.While, Mokhtar (2004) recovered H. pylori from the examined ice cream samples in lower incidence (2%).Presence of H. pylori in the examined samples might be due to incorporation of contaminated milk in manufacturing of ice cream or due to poor sanitation during preparation and storage of these products.H. pylori isolates were confirmed by PCR (Figure 1).
Besides this most well-known member i.e., H. pylori, more than 20 other Helicobacter spp.have been identified and several of them are associated with various chronic digestive diseases (Zhou et al., 2011).H. pullorum has been related to diarrhea, gastroenteritis and liver disease in humans (Ceelen et al., 2005).Also, H. cinaedi causes bacteremia, cellulitis, septicemia and enteritis in immunocompromised, immunocompetent and healthy persons (Taniguchi et al., 2016).
H. pylori colonizes in the stomach of more than 50% the world's population.Majority of these bacteria occur in the mucus layer of the stomach and may act as a reservoir of infection for the underlying gastric epithelium (Dunne et al., 2014).Moreover, it is widely accepted that the bacteria in contact with epithelial cells initiate disease (Klerk et al., 2016).WHO has assorted H. pylori as a class I carcinogen and its elimination with antibiotic combinations has been notified to be profitable in preventing gastric sickness particularly cancer (IARC, 1994) before increasing antibiotic resistance problem which generated an obstacle in treatment of H. pyloriassociated diseases by usual therapies (Murali et al., 2014).
In this study, we investigated the anti-H.pylori effect of essential oil extract of mastic gum in lab prepared ice cream.First, we had to determine the anti-H.pylori activity of the obtained mastic oil through detecting the inhibitory zone which was found to be 19 mm, then the MIC and MBC were estimated and they were 0.4 and 0.8% v/v, respectively.It was difficult to compare the MIC and MBC with other studies as different mastic substances and methodologies have been used by different researchers yielding various results (Tassou and Nychas, 1995).Huwez et al. (1998) observed that crude mastic gum could kill H. pylori at a concentration of 0.06 mg/mL.While in another study, the mastic extracts hardly exerted any antimicrobial activity (Thuille et al., 2003).Also, Marone et al. (2001) who found that 50% and 90% of H. pylori strains were inhibited at mastic gum concentration of 125 µg/mL and 500 µg/mL, respectively.While, Miyamoto et al. (2014) found that from the identified 20 chemical compositions of mastic gum essential oil, an (E)-methyl isoeugenol exhibited potent antibacterial activity against four H. pylori strains including clarithromycin and metronidazole resistant strain.Sharifi and Hazell (2011) reported that the MIC values of essential oil of the mastic gum examined ranged 500-1000 mg/mL and its major constituent was α-pinene.
According to our knowledge, there is a study concerning antimicrobial effect of mastic oil against E. coli O157 in yoghurt (Pagiataki et al., 2013).However, this is the first study discussing the anti-H.pylori effect of essential oil extracted from mastic gum in ice cream.In ice cream samples stored at domestic freezer temperature (-5°C), the added essential mastic gum oil (0.8 and 1.6% v/v) suppressed quickly the growth of the inoculated H. pylori (approximately 1-log and 1.2 log, respectively) within the hours of hardening.The most bactericidal action was observed after third day of storage causing about 4-logs decrease in the load of H. pylori with the reduction of 99.2% for the sample treated with 0.8% v/v of mastic essential oil, while it was at the second day of storage with reduction of 99.7% in ice cream sample treated with 1.6% v/v of mastic gum essential oil.H. pylori couldn't be counted (reaching 100% reduction) or isolated from samples at the first week versus the third day of storage for samples treated with mastic essential oil at concentration of 0.8 and 1.6 % v/v, respectively, while at    the same time, its count was 8.5x10 3 and 7.9x10 4 CFU/mL in control samples, respectively (Table 2).
From data showed in Table 3, it was found that the high initial population of H. pylori (inoculum 10 6 CFU/gm) in samples stored at -20 °C was reduced by about 2-logs and more than 2-logs in samples treated with 0.8 and 1.6% v/v essential oil of mastic gum, respectively during the hours of hardening.The most drastic effect on H. pylori was observed in ice cream samples held at -20°C at the second day of storage for both concentrations 0.8 and 1.6% v/v (about 99 and 99.6 reduction %, respectively).We failed to count or isolate H. pylori at the third day of storage from samples contained 0.8 and 1.6% v/v mastic oil, respectively, where it was found in control sample with a count of 4x10 3 CFU/mL at the same time.Also in these samples, it was noticed that there is slower dimension rate of H. pylori load as compared to those stored at -8°C also 100% reduction in H. pylori couldn't be reached in samples with 1.6% v/v mastic oil before samples with 0.8% v/v mastic oil and stored at -20°C as expected.We couldn't find an explanation clarifying this behavior of mastic essential oil under -20°C.The pronounced effect of mastic oil against microarobic bacteria could be related to its enhanced activity under lower oxygen tensions (Gkogka et al., 2013).
From food safety point of view, Solnick et al. (2001) stated that the minimum infectious dose of H. pylori is 10 4 CFU and it was found that 0.8 and 1.6% mastic oil concentrations achieved reduction in H. pylori under 10 4 CFU at the second and first day of storage of ice cream samples stored at -5°C, respectively, while in samples stored at -20°C, this reduction under 10 4 CFU was accomplished after the first day of storage and after hardening time for the two concentrations, respectively.
From the therapeutic point of view, ice cream with mastic gum essential oil can play role as a functional food as its inclusion in the diet can be helpful, not only in patients with clinical symptoms but also in case of asymptomatic patients as it could reduce the risk, as well as the development of an unfavourable outcome of the infection.

CONCLUSION
Food safety regulations as well as quality standards including good manufacturing practices (GMPs) and hazard analysis and critical control points (HACCP) should be more concerned in Egyptian food in order to control contamination and proliferation of pathogenic bacteria.The present study shows that mastic oil at the tested concentrations has spectacular bactericidal effect against H. pylori inoculated in ice cream.Ice cream being so delicious food could be modified to a functional food by addition of essential oil of mastic gum and used as a therapy and introduced to the diet of patients to help in eradication of H. pylori from patients' stomach.

Figure 1 .
Figure 1.Confirmation of H. pylori isolates by PCR reaction.Lane L: Ladder, Lane +ve: positive control, Lane -ve: negative control.Lane 1, 2, 3: Helcobacter isolates obtained from ice cream samples, Lane 2 and 3 are H. pylori isolates and amplified at 417-bp fragment of the flanking region of H. pylori ureC gene.

Table 1 .
Incidence of different Helicobacter spp. in the examined ice cream samples

Table 2 .
Effect of different concentrations of essential oil extracted from mastic gum on H. pylori in lab prepared ice cream stored at freezing temperature (-5°C)

Table 3 .
Effect of different concentrations of essential oil extracted from mastic gum on H. pylori in lab prepared ice cream stored at domestic deep freezing temperature (-20°C)