Application of some insensitive probiotic lactic acid bacteria and ginger as functional dairy products

The use of dairy-based functional foods has increased markedly over the last few years. Ginger is believed to exert a wide range of therapeutic properties. This study aimed to evaluate probiotic properties of twenty Lactic acid bacteria to apply in functional fermented milk fortified with fresh ginger juice to increase the therapeutic and nutritional effects of the product. Ginger juice was prepared from ginger rhizomes through sorting, washing, peeling, crushing and crude juice extraction. It was assayed for antibacterial activity by minimal inhibitory concentration against antibiotic resistant pathogenic bacteria (E. coli BA 12296, Bacillus subtilis DB100, Klebsiella pneumoniaa ATCC12296, Salmonella senftenberg ATCC 8400, Staphylococcus aureus NCTC 10788, Staphylococcus epidermidis ATCC 35984). Total phenol, total flavonoids and antioxidant activity were determined, ginger juice showed an antibacterial activity and antioxidant activity. Twelve strains out of twenty were resistant to bile salts concentrations (0.2, 0.3 and 0.4 w/v), to acid conditions (pH 2.0, 3.0 and 4.0) and were able to grow in the presence of 0.2 and 0.4 w/v of phenol. Seven strains (Lactobacillus delbrueckii subsp. delbrueckii KT615, Lb. brevis KP653, Lb. delbrueckii subsp. lactis KP645, Lb. plantarum KP623, Lb. paracasei subsp. tolerans WT631, Enterococcus faecalis BM711, Enterococcus faecium BT734) had the ability to adhere to rabbit intestinal epithelial cells in-vitro, and were resistant to ginger juice. Ginger juice concentration showed indirect relationship with the milk coagulation time and direct with syneresis of fermented milk. It was showed that used 2% of ginger juice concentrations was the best results on fermented milk products.


Introduction
Functional foods are recognized as having physiological benefits as well those of basic nutrition which called nutraceuticals (Bigliardi and Galati 2013).Nutraceuticals are a natural product sold in dosage form (capsules, tablets, powders, etc).Functional dairy products are the healthy food products especially probiotics products which were defined as products have a live microorganism that when treated in suitable amounts give a health benefit to the host (FAO/WHO 2002).In recent years, different studies support the importance of probiotics as apart of healthy diet for humans and animals and as a way to provide a natural, safe and effective barrier against microbial infections (Markowiak, and Śliżewska, 2017;Kerry et al. 2018;Vijayaram and Kannan 2018).Lactic acid bacteria (LAB) are non-pathogenic bacteria that belong to the major group of probiotic natural microbiota in the gastrointestinal tract (GIT) and preserve an efficient balance between useful and harmful bacteria.
Recent studies have shown the role of lactic acid bacteria as a probiotic and its functional and health properties.Ayyash et al. (2018) showed in-vitro the health-promoting effects (anticancer activity, α-amylase and α-glucosidase inhibitions, ACE-inhibition, antioxidant and proteolytic activity) of camel milk fermented with three probiotic strains of Lactobacillus spp.were isolated from camel milk.Also, the effects of probiotic LAB supplementation on inflammation and oxidative stress was studied in many studies due to the probiotic role in immune system modulation and the anti-inflammatory response (Plaza-Díaz et al. 2017;Badehnoosh et al. 2018).In another study of nine probiotic LAB strains were isolated from different sources and identified have high antagonistic activity.There is one isolate Lactococcus lactic subsp.lactis BO37 has manifested the highest cholesterol removal ability in vitro (Shehata et al. 2016).
Ginger rhizome has been used as a medicinal herb due to its high content of antioxidants and anti-inflammatory properties.In the last decades, ginger has been used in the medicinal application against pathogenic microorganisms.It was reported that ginger has antipyretic, analgesic and anti-cancer properties (Du et al. 2018;Mathew 2018).In addition, the rhizome of ginger has many nutrients as fats, carbohydrates, protein, fiber and water.It contains volatile component which confers the unique flavor of the spice.It has been mentioned that: "Round amongst them (the righteous in paradise) is passed vessels of silver and goblets made of glass a cup, the admixture of which is ginger" (The Holy Quran 76:15-17).Ginger is a common spice which contains bioactive constituents such as zingerone, shogaols, paradol and gingerols.It has been extensively studied for its pharmacological and biological activities (Foster 2011;Singh et al. 2018).Due to the low toxicity of ginger, its contents of bioactive components and its broad spectrum of biological and pharmacological applications, has been increasingly used (Aly et al. 2013).
Antioxidants were defined as "substance that in small quantities are able to prevent or greatly retard the oxidation of easily oxidizable nutrients such as fats" (Skibsted 2010).It can prevent oxidative damage to food during processing, storage and preparation of meals.Antioxidants may accordingly help the development of more healthy food with low levels of lipid and protein oxidation products.Also, antioxidants have more compounds able to scavenge free radicals such as peroxide, hydroperoxide or lipid peroxyl and thus inhibit the oxidative mechanisms that lead to degenerative diseases (Valko et al. 2006;Al-Rimawi et al. 2017).Ginger has antioxidant activity by the antioxidant components analyzed such as polyphenols, vitamin C, flavonoids and tannins they have displayed strong antioxidant activity in-vitro (Atashak 2014).
Ginger also showed antimicrobial activity against pathogenic bacteria like some Salmonella strains, Staphylococcus aureus, Yersinia enterocolitica, Escherichia coli and Listria monocytogenes, Saccharomyces cerevisia and filamentous fungi e,g.Aspergillus (Aruna et al. 2014;El-Khalek et al. 2016).Therefore, many studies have been carried by several investigators suggested ginger as a preservitaive and medicatication supplement against microbial spoilage of food and for topical antifungal or antibacterial treatment (Policegoudra et al. 2007;Hasan et al. 2012).
Due to shortage of research concerning probiotic fermented dairy products fortified with fresh ginger juice due to its antibacterial properties, this work aimed to: 1-use the ginger juice in dairy products to enhance their functional and nutritional properties, 2-selection of insensitive probiotic LAB as a supplement, 3-study the sensory evaluation and properties of the product, and 4-produce innovate new fermented dairy products.

Lactic acid bacteria strains
Twenty strains of LAB (Table 1) have antimicrobial activity against culture of enteropathogenic E. coli obtained from the culture collection of NIZO (Food Research, Ede, The Netherlands) were kindly supplied by culture collection of Faculty of Agriculture Saba Basha, Alexandria University (FABA).

Assessment of potential probiotic LAB
Acid resistance and bile salt tolerance were assayed using the method of Charteris et al., (1998) and Zinedine and Faid (2007) with some modification.The strains were incubated at 37°C / 6 h, then optical density was measured at 650 nm (O.D 650 ) using a Spectrophotometer (Apel-pD-303UV Spectrophotometer, Japan).Strains were inoculated (10% v/v) into MRS broth previously adjusted to pH (2.0, 3.0 and 4.0 ±0.1) with HCl.MRS broth containing bile salts was prepared by the addition of 0.2, 0.3 and 0.4% (w/v) bile salts (Biolife, Milano, Italy).Overnight cultures were inoculated 10% (v/v) into MRS broth and incubated at 37°C.Bacterial growth was followed for 6 h by measuring the optical density at 650 nm.
MRS broth and M17 were modified with 0.2 to 0.4% phenol to determine the phenol tolerance of the isolates according to the method described by Aswathy et al. (2008).Inoculated cultures were incubated at 37 ºC for 24 h.Absorbance (as a function of growth) was measured at 600 nm after 24 h.This experiment was performed in triplicates.

Adhesion to rabbit intestinal epithelial cells Adherence assay
Epithelial cells were obtained from the small intestines of rabbits (2.5±0.25 Kg) as previously described by Alnaqdy et al. (2005).The small intestine of fresh slaughtered healthy rabbits was removed and chilled in cold physiological saline solution and brought to the laboratory within 30 min.Seven cm sections of freshly collected duodenum from rabbit was slit open, washed with cold PBS pH 7.2 (4 o C) and incubated in a buffer containing 10 mM EDTA pH 6.8 for 20 min.The section was then rinsed twice with phosphate buffer saline (PBS) to remove the EDTA then placed in 5 ml of cold PBS.Epithelial cells were dislodged by rubbing the intestine with a sterile syringe plunger and the epithelial-rich supernatant (which was identified from other cell fractions, such as leukocytes, on the basis of cell size and morphology) was removed with a sterile Pasteur pipette, pelleted by centrifugation at 100 xg for 10 min, washed twice with PBS (100 xg for 5 min) and then resuspended in 2 ml PBS.The number of epithelial cells was adjusted to 10 6 cell/ml using a haemocytometer.
One ml containing 1 x 10 8 cells of LAB strains were mixed with equal volume of epithelial cells at 10 6 cells/ml and incubated for 1 h at 37ºC in a shaker water bath (Kottermann, D.3162 Germany).Epithelial cells were then pelleted by centrifugation at 100 x g for 10 min, washed with PBS to remove any unattached bacteria and resuspended in 2 ml PBS.Microscopic slides were prepared and stained with crystal violet.The numbers of Lactobacillus cells attached to single epithelial cells were counted under the oil immersion using a light microscopy (Optika microscopes, Italy).E. coli was processed in the same manner as a control.This experiment was performed once counting the adhered bacteria to 50 epithelial cells.

Ginger juice extract
Ginger juice was prepared according to the method of Akhani et al. (2004) in which fresh rhizomes of ginger (2Kg) obtained from local markets in Alexandria, Egypt were crushed and squeezed in muslin / cotton cloth to obtain the juice, which was stored in the refrigerator at 2-7° C in a well-closed dark glass container.

Chemical analysis of ginger juice
Total soluble solids, ash content and pH were determined as described by Official Methods of Analysis of AOAC International (2016).

Total phenol contents
Total phenolic contents of ginger juice (ml) were determined using Folin-Ciocalteu reagent according to the method described by Stoilova et al. (2007).

Total flavonoids determination
The aluminum chloride colorimetric assay method was used to determine total flavonoid content of ginger juice (ml) and use catechol as an equivalent according to the method described by Sakanaka et al. (2005).

Free radical scavenging determination using DPPH method
Antioxidant activities of ginger juice was evaluated through free radical scavenging effect using 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical according to the method proposed by Akowuah et al. (2005).

Antimicrobial activity of ginger against the pathogenic bacteria
The minimal inhibition concentration (MIC) values of the ginger juice against pathogenic bacteria was determined after 48 h of incubation at 37 o C using microdilution method (Wang et al. 2010).For the determination of minimal growth concentration (MGC), a portion of liquid (5 μl) from each plate well that exhibited no growth were taken and then incubated at 37 o C for 24 h (Kang et al. 2011).

Antagonistic effect of ginger juice on selected probiotic strains
The disc diffusion assay (Pessoa et al. 2017) was used for detection of antagonistic effect of filtered ginger juice against 12 selected probiotic LAB strains.Loop full growths from probiotic bacterial strains were inoculated into MRS broth, incubated at 37 °C for 18 h containing 10 8 CFU / ml.Cotton swab was dipped into adjustment suspension and streak the entire MRS Agar media surface of plates and the plates were left for 15 min at room temperature to dry.A half cm wide filter paper discs were sterilized and 10 μl of the ginger juice was placed on the disc.All of plate tested was incubated at 37°C for overnight.After 24 h of incubation, ginger juice was noted for zone of inhibition for all probiotic strains.The diameters of the zone of inhibitions were measured by measuring scale in millimeter (mm).

Preparation of ginger yoghurt
Pasteurized skim milk (12% SNF) was warmed to 42 o C and cultured with 1-2% of the common yoghurt cultures Yo-Mix ® 200 DCU (Danisco, USA) with different concentration of ginger juice (0, 1, 2, 5, 10 and 15%).The mix was distributed in cups and incubated at 42 o C for 3 h, then cooled and stored at 4 o C (Tamime and Robinson 1985).

Syneresis
One hundred grams of ginger yoghurt samples with different concentration of ginger juice (0, 1, 2, 5, 10 and15 %) were placed on a filter paper resting on a top of a funnel.After 2 h of drainage at 7 o C, the quantity of whey out of the total weight (100 g) of the yoghurt collected in 50 mL graduated cylinder was used as an index of syneresis (Srisuvor et al. 2013).

Sensory evaluation of ginger probiotic fermented milk
Sensory evaluation was performed by overall quality score of the conventional fermented milk according to Majchrzak et al. (2010) and Allam et al. (2017).It was conducted by a trained panel of 10 assessors (8 men and 2 women, aged from 27 to 58 years) at Faculty of Agriculture, Saba Basha Alexandria University.The method describes all sensation perceived when evaluating a sample by different categories as appearance, aroma, flavor/taste, texture and after taste.Every category is described by certain descriptors, which are discussed and agreed upon the definitions by the panelists during evaluation.The averages of sensory evaluation data with standard deviations were determined.

Statistical analysis
Statistical analysis was performed using Analytical Software SPSS® 13.0 (Statistical Package for the Social Sciences) (2005).Values are mean of ±SD (standard deviation) of three replicates.Differences were considered significant at P < 0.05.

Chemical analysis of ginger juice
Chemical composition of ginger juice is exhibited in Table (3).Results showed that ginger juice contains 79.8% moisture, 20.5 % TSS and 1.17% Ash.These results almost agreed with Zadeh et al. (2014).

Total phenolic and flavonoids content
Antioxidants contents, phenolic and flavonoids, of ginger juice are presented in Table (3).Results clearly indicated that ginger juice has high content of total phenols with mean a value of 161.074 mg GAE/100 g juice which higher than that of results obtained by Maizura et al. (2011) (101.56 mg GAE/100g ginger extract).Flavonoid content of ginger juice was 27.317 mg catechol /g.Pawar et al. (2011) found that flavonoid content in ginger extracts ranged from 1.3 to 3.8 mg quercetin/g.

Antioxidant capacity
Accordingly, antioxidant potentials of ginger juice to scavenge DPPH is presented in Table (3), This result in agreement with Mošovská et al. (2015), who mentioned that ginger extract is a good source of polyphenolic compounds including gingerols, shogaols, paradols and gingerdions.Our results suggested that demonstrating of that ginger juice can be performed as a natural antioxidant in the functional food and pharmaceutical industries.This may be refered to the phenolic compounds of ginger juice which of considerable interest and are increasingly becoming a subject of intensive research due to their bioactive properties (Ignat and Popa 2011;Tohma et al. 2017).So, the support of fermented milk products with ginger can increase the functional properties of the product.Twelve selected probiotic LAB strains are exhibited in Table ( 4).The results showed the antagonistic effect of ginger against five tested probiotics (Lb.fermentum KT642, Enterococcus faecium KT724, Leuconostoc oenos EP6494, Lb. casei BP631, Enterococcus durans BT611), while seven strains were insensitive to ginger juice.

Antimicrobial activity of ginger against the pathogenic bacteria
The data presented in Table 5 shows that ginger juice exhibited high antibacterial activity against all tested pathogenic bacteria.It was more effective against Staphylococcus aureus NCTC 10788 (MIC 100 µl/ml and MGC 150 µl/ml), as compared to other strains.Bacillus subtilis DB100 showed less sensitivity to ginger juice (MIC 200 µl/ml and MGC 250 µl/ml).These results agreed with those of (Panpatil et al. 2013;Islam et al. 2014).

Properties of ginger yoghurt
The effect of adding different concentration of ginger juice to produce ginger yoghurt and its properties showed in Table 6.The syneresis increased with high percentage of ginger juice and vice versa coagulation time.Once again the results in agreement with those reported by Su et al. (2009) which showed that ginger with milk clotting activity has been traditionally used in the preparation of ginger milk curd in China.The results also indicated that yoghurt supplemented with 2% ginger juice produced acceptable ginger yoghurt with excellent flavor.While by increasing ginger juice concentration from 5 to 15% resulted in an unacceptable flavor due to an oily substance called gingerol (Ajav and Ogunlade 2014).-The evaluation of the intensity of the descriptors is done using a 10 unit scale.
-Overall sensory quality = overall impression of the products.

Conclusion
Like the Father of Medicine Hippocrates, said "Let your food be your medicine and your medicine be your food" which clearly confirm a linkage between diet and health for human.We have to select healthy food have a functional and medicinal properties, antioxidant, antimicrobial, and have good sensory properties in the same time .
There is an ideal correlation between total phenol content and antioxidant activity that supports the idea of phenols as contributor of the antioxidant power of ginger juice when support the functional dairy product with it.
The results showed that the addition of ginger juice in milk at concentrations ranging from 1 to 15% (v/v) reduced the coagulation time and pH of different yoghurt samples and increased the percentage of syneresis.Also, the supplementation of 2% ginger juice was observed to be optimum between all ginger juice concentrations added to milk for yogurt processing.
On another hand, the addition of selected probiotics strains to fermented milk with 2% ginger juice the obtained results showed that treatment containing Lb. paracasei subsp.tolerans WT631, Lb. brevis KP653 and Lb.del.subsp.lactis KP645 probiotic strains scored high levels of overall intensity.
In addition to that, there is little studies about the addition of fresh ginger juice to probiotic fermented milk and yoghurt to enhance its functional properties so, this study recommended to use fresh ginger juice as a fortified in fermented milk and studied the medical properties of products.

Table 1
Lactic acid bacteria strains

Table 2
Assessment of probiotic criteria of LAB strains *Number of LAB strains adhered to epithelial cell (total of 50 epithelial cell were studied) *: Data are represented in means of (3n) ±SD; ND, not determined

Table 3
Chemical composition, antioxidants contents and potentials of ginger juice Total phenolic was expressed as gallic acid equivalents (GAE) mg/ g sample.**Total flavonoids were expressed as mg catechol/g sample.***IC50 (mg/mL): Inhibitory concentration at which 50% of DPPH radicals are scavenged.

Table 4
The antagonistic effect of ginger juice against selected probiotic strains

Table 5
Minimal inhibitory concentration of ginger juice (µl/ml) against the pathogenic bacteria

Table 6
Properties of ginger yoghurt with different concentration

Table 7
Sensory evaluation of probiotic ginger fermented milk -Averages ± Standard deviation (SD) of three replicates.