THE EFFECT OF CAMEL MILK WHEY PROTEINS ON LACTIC ACID BACTERIA ISOLATED FROM CAMEL AND COW MILKS IN EGYPT: A COMPARATIVE STUDY

Mariam Mohammed 1 , Marwa H.El-Gendy 1 , Aida S. Salem 2 and Wafai Z. A. Mikhail 3 . 1. Animal Production Division, Desert Research Center, Cairo, Egypt. 2. Dairy Technology Department, Animal Production Research Institute, Agricultural Research Center, Giza, Egypt. 3. Department of Natural Resources,Instituteof African Research and Studies, Cairo University, Cairo, Egypt. ...................................................................................................................... Manuscript Info Abstract ......................... ........................................................................ Manuscript History

987 milk and are rich in protective components include lysozyme, lactoferrin, lactoperoxidase (LP) and peptidoglycan recognition protein (PGRP) which only detected in camel's milk (Singh et al., 2006;Kappelr et al., 2004), IGA and IGg immunoglobulin's that are compatible with human ones and provide effective defense against several viral and bacterial pathogens (Khitam, 2003). Camel's milk consumption may also be helpful in reducing the nutritional deficiencies and morbidities in adult community (Agrawal, et al., 2005;Singh et al., 2009).
Lactic acid bacteria (LAB) are considered as a Gram positive, non-spore forming, catalase-negative and acid tolerant microorganisms that produce lactic acid as the primary end-produced during carbohydrate fermentation (Carr et al., 2002). (LAB) are one of the most important beneficial microbiota in camel milk, which is vitality of an important source for various dairy technology (Khedid, et al., 2009). LAB are among the important groups of bacteria providing health benefits to human, animal and plant (Song, et al., 2012). Using LAB in food is one of the ancient known food preserving techniques. LABs are widely spread. It was found in many different food products (dairy, meat, beverages and vegetables), but they are also present in the mouth, digestive system and vagina of mammals (Hayek and Ibrahim, 2013). Also LAB plays an important role in the food industry, as they are used for health improvement, production of macromolecules, enzymes and metabolites (Pfeiler and Kleinhammer, 2007).
Bacteria in general, require an appropriated biochemical and environment to grow and express normal metabolic activities. There are many factors that affect the growth of LAB strains, including those related to intrinsic the food itself (water activity; pH value; nutrient content; antimicrobial substances and mechanical barriers to microbial invasion; redox potential.), including extrinsic related to the environment (the temperature of storage; the atmosphere surrounding the food). Also, implicit factors are the factors related to the microorganisms themselves and processing factors. (Hamad, 2012,Hayek andIbrahim, 2013).
Biochemical conditions including stimulatory, preferred, and essential nutrients have significant impact on the growth and studied throughout the world and it approved that camel's milk contains several antimicrobial agents including lactoferrin, lactoperoxidase, lysozyme and PGRP (Reiter, 1985;Renner, et al., 1989;Kappler, et al., 2004;) that found with high concentration in whey of camel's milk. The effect of this antimicrobial agent against pathogenic bacteria such as Escherichia coli and Rotavirus has been studied in several investigations but its effect on LAB is not clear till now. Furthermore, isolating and classifying this bacterial group from camel's milk and selecting them as starter cultures are important steps for the better use of camel's milk.
Thus, the aim of this investigation is to evaluate the effect of whey camel's milk (that is rich in antimicrobial proteins) on the behavior of lactic acid bacteria isolated from camel and cow's milk in parallel.

Materials and Methods:-
Sampling:-Total 14 samples of camel's milk and 5 samples of raw cow's milk and 2 samples of zabady (a traditional fermented product from cow milk) were collected from different regions in Egypt and Sudan. Camel's milk samples were collected from several regions in Egypt including; King Maryout (5 samples) and El Amrya (2 samples) at the Alexandria governorate; Matrouh (2 samples) andEl Omyed (2 samples)at the Marsa Matrouh governorate; Halayeb and Shalteen (1samples) at the New Valley governorate and one sample from north of Sudan. Samples of camel milk were collected at the same season (winter-spring for year 2015-2016) and milking was performed randomly during different periods ranging from one day to 3 months after parturition whereas, cow milk samples were collected from Alexandria governorate at the winter season. All samples were immediately cooled and transported to the laboratory in an icebox (4 °C) and analyzed for the content of LAB on the arrival.

Isolation of Lactic Acid Bacteria:-
Thirty ml of each milk sample were incubated at 30˚C (for mesophilic LAB), 42˚C (for thermophilic LAB) and 37˚C (Both LAB) until coagulation for 24 hours to facilitate their isolation. The cultures were streaked on different media included M17 agar (Biolife Italy) for lactococci, MRS agar (de Man et al., 1960) (for lactobacilli) at 30˚C, 37˚C and 42˚C respectively, ST agar for Str. thermophilus and SF agar for enterococci at 42˚C for two days. Representative colonies were collected according to their shape and colour. Isolates were purified by streaking two times on the respective isolation medium and temperature.The isolation of Bifidobacteria spp. were performed by enumerating coagulated samples on modified MRS broth ( MRS supplemented with sodium acetate (10gm/L), 0.5g /L L.cyctein and pH was adjusted to 5.4 by acetic acid) followed by streaking at the same medium and isolated as described before.
988 Identification of isolates:-Phenotypic identification:-All isolates were microscopically examined for Gram stain reaction, cell morphology and cellular arrangement. Catalase activity was examined by adding drop of 3 percent hydrogen peroxide on a clean microscopic slide. A visible amount of bacterial growth was added with the inoculating loop. Both were mixed and observed for gas bubble production. Only Gram-positive and catalase-negative isolates were identified at species level.The production of carbon dioxide from glucose was carried out as follows, 0.5 ml of the purified isolate was inoculated in 2 ml medium (at 40 -45˚C) after that 2 ml water agar (1.5%) was added as an agar plug, incubated at 37˚C and observed for gas production after 24 -48 h. A control was carried out using positive culture for carbon dioxide production "Saccharomyces lactis N.C.Y.C. 571".

Growth at 45˚C and at 10˚C:-
The ability of isolates to grow at 45˚C or at 10˚C were carried out as follows, each isolate was inoculated in broth medium; MRS of lactobacilli and M17 for lactococci (1%) and then incubated at 45˚C or at 10˚C for 48 h. The growth was observed in the medium; the control was an un-inoculated tube and incubated under same condition.
Growth in the presence of 6.5% salt?:-This test was limited to the lactococci only; salt tolerance was assessed after 3 days of incubation at 6.5% NaCl in M17 broth.

Growth in SF medium:-
This test was limited to the lactococci, the growth of strains was assessed after 48 h of incubation in SF medium at 42˚C.

Molecular Identification of Lactic Acid Bacteria Species:-
The isolates that isolated from fresh samples only were subjected to their genotypic characterization using BOX and ERIC PCRs as described below:

Reference strains and cultivation:-
The reference strains were obtained from Institute National de Recherche Agronomique (INRA), Center National de Recherches Zootechnique Jouy-en Josas, France (CNRZ), American Type Culture Collection (ATCC) and (DSM) Deutsche Sammlung von Mikroorganismen. These strains had been previously characterized on the basis of microscopic examination, Gram staining and catalase reaction. Stock cultures were maintained at -40ºC in reconstituted skimmed milk plus 15 % glycerol and working cultures will be prepared from frozen stocks through MRS or M17 broth.

DNA Extraction:-
For DNA extraction, 1ml of an overnight culture was added to a 1.5ml microcentrifuge tube and Centrifuged at 13,000-16,000 × g for 2 minutes to pellet the cells. The supernatant was removed. DNA was extracted from cells pellet using Wizard Genomic DNA Purification Kit (Promega, Madison, USA) as described by its manufacture.

Repetitive -Polymerase chain reaction:-
The DNA concentration of each sample was adjusted to 25 ng/µl in a 25 µl PCR mixture. Amplification was performed in a 25 µl reaction as described by Mohammed et al. (2009) and Kŕĭžov ă et al. (2008). The molecular sizes of the amplified DNA fragments were estimated by comparison to a 100 bp DNA ladder (Promega, Madison, USA) and were photographed using polaroid film. The Rep profiles were processed using the Gel ComparІІ version 5.00 software (Applied Maths, Kortrijk, Belgium). Similarity coefficients for pairs of tracks were calculated by using Pearson product moment correlation coefficient and strains were grouped by using the unweighted pair group method with arithmetic average (UPGMA).

Preparation of camel milk whey:-
Ten ml of camel milk was centrifuged at 5000xg for 30 min at 10˚C to remove fat components. Whey was obtained after acid precipitation of casein at pH 4.6 by adding 1ml of 10% (v/v) acetic acid and incubated at 37˚C for 10 min. Following it was neutralized 1/10 vol of sodium acetate and centrifuged at 10.000 xg for 5 min at 4˚C. The supernatant was then dialyzed for 48hrs and stored at 4˚C until used.

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Determination of total whey proteins:-Twenty-five ml of whey (that was naturalized previously by 1/10 vol. of 1M sodium acetate) was added in a breaker. Then, 1ml potassium oxalate solution (28%) and 0.25ml phenolphthalein (2%) was added into the sample. After mixing, the solution was titrated against sodium hydroxide (N/7) until the pink color appeared and then 5ml. of neutralized formalin solution (40%) was added to disappear the pink color followed by second titration against NaOH (N/7) until a faint pink color appears again and the second reading was represented as protein%.
Resistance to whey of camel milk:-Two hundred millimeter of camel milk whey (that included 12.2gm of soluble whey protein) was added to 800ml MRS media. Then, 1% of lactic acid bacteria strains were inoculated in media supplemented with whey. The optical density was determined at 650nm during 3, 6 and 24 hours compared with control (supplemented media without bacterial inoculation).

Identification of Lactic acid bacteria:-
The isolation of LAB from natural source have always been the most powerful means for obtaining useful and genetically-stable strains for industry important products

Pre-identification using morphological tests and biochemical reactions:-
In the present study, fifty-eight isolates that including 41 isolates obtained from camel milk and 17 isolates were obtained from cow milk. Pre-identification of isolates of different camel's milk and cow's milk samples is illustrated in Tables (1-2

20CM
KM Pre-identification using morphological tests and biochemical reactions of different camel's milk and cow's milk samples is showed in Tables (3).

Identification by rep-PCR fingerprints:-
Fifty-eight isolates were further characterized to species and subspecies level using Rep-PCR technique using BOXA1R primer for LAB. Identification was based on similarity coefficients for pairs of tracks calculated by using Pearson correlation coefficient and strains were grouped by using the unweighted pair group method with arithmetic average. Based on phenotypic and biochemical characteristics, the pre-identified isolates that belonging to group A, group B and group C for Lactobacilli, Enterococcus, Lactococcus and Streptococcus for lactococci and Bifidobacteria were subjected to genotypic identification using Rep-PCR as shown in Figures (1-9).   Figure (1) shows that seven isolates obtained from camel milk and one isolate obtained from cow milk were identified as Lactobacillus plantarum (Lb. plantarum) with similarity level 75% and 50% respectively comparing with CNRZ 5050 as a reference stain. On the other hand, two isolates obtained from camel milk and three isolates obtained from cow milk that were pre-identified as group A previously, were identified as Lactobacillus acidophilus (Lb. acidophilus) by Rep-PCR fingerprints at 75% and 60% similarity level respectively as shown in Figure (2), as well as, two isolates obtained from both camel and cow milks were identified as Lactobacillus delbrueckii subsp lactis (Lb. del. lactis) at similarity level 90% comparing with CNRZ 250 as a reference strain (Figure 3). Belonging to group C lactobacilli, five isolates from camel milk and one isolate from cow milk were identified as Lactobacillus fermentum (Lb. fermentum) with similarity level 85% and 80% respectively comparing with CNRZ64 as a reference strain (Figure4).
As illustrated in Figure (5) two isolates from camel milk and three isolates from cow milk were identified as Lactobacillus rhamnosus(Lb. rhmnosus) with similarity level 80% comparing with the reference strain.
Twenty-nine from lactic acid cocci that were pre-identified previously as Lactococcus, Enterococcus and Streptococcus were distinguished by genotypic identification. All Streptococcus isolates (2 obtained from camel milk and one from 994 cow milk) were clearly identified as Streptococcus thermophiles (Str. thermophiles) with a similarity level 60% comparing with DMS 20259 as a reference strain ( Figure 6). Whereas six isolates from camel milk and two isolates obtained from cow milk were identified as Lactococcus lactis subsp. lactis (Lc. lactis.lactis) with similarity level 90% compared with ES005 as a reference strain and two isolates obtained from camel milk only were identified as Lactococcus lactis subsp.cremoris (Lc. lactis cremoris) and Lactococcus garvieae (Lc. garvieae) that were detected for the first time in camel milk comparing with Z 105T and CNRZ 219 as a reference strains respectively as described in Figure (7).
Among Enterococcus twelve isolates obtained from camel milk and four isolates obtained from cow milk were identified as Enterococcus faecium (En. faecium) using Rep-PCR amplification with similarity level 65% comparing with CNRZ 131 as a reference stain (Figure 8). On the other hand, one isolate was identified as Bifidobacterium animalis (B. animalis) and represented as a minor species of LAB in camel milk (Figure 9). This result was in agreement with Savadogo et al.,(2004) who found detected Bifidobacterium species in traditional fermented camel milk and it represented 10% of LAB isolated from samples studied.
The percentage of LAB strains from camel and cow milks were illustrated at Table (4). The obtained results revealed that Lc. lactis subsp. cremoris and Lc. garvieae that detected at the first time in camel milk were found in samples collected from Marsa matrouh governorate that located near the Mediterranean sea. The presence of this species in this sample of camel milk may be due to its high resistance to salt concentration as Lc garvieae always found in marine environment.
In this study the biodiversity of LAB obtained from camel milk from different regions in Egypt showed that noticed belonging to Lactobacillus acidophilus and Lb. del. lactis that could not be distinguished in camel milk samples obtained from north Egypt and found in samples collected from Halayeb and Shalateen and north Sudan, and this also was in agreement with Estiphano et al., (2016) who found the same species in raw camel milk from Ethiopia. These results may be due to the climate conditions in Sudan and east of Africa countries where high temperature encourages thermophilic bacteria to grow and become dominant LAB species in these regions.

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Our results regarding to the biodiversity of LAB isolated from raw camel milk were in agreement with many researchers who considered theses species of LAB isolated from raw camel's and cow milks and its traditional fermented dairy products. ( The ability to resist whey of camel milk:-All LAB strains isolated from both camel and cow milks were tested to grow at constant concentration of whey camel's milk Figure (10). interprets the averages of optical density values of bacterial growth at 650nm. The obtained results revealed that all LABS that isolated from camel's milk showed a good ability to grow at the 20% of camel milk whey during 24hours compared with those isolated from cow milk. A gradual increase of bacterial growth was observed through 3,6 and 24 hours of incubation regarding LAB strains isolated from camel milk , whereas, a sharp decline was noticed of the growth of strains isolated from cow milk when examined to grow in cultures supplemented with whey.  Figure (11)illustrated the comparison of the bacterial growth between those isolated from camel and cow milks when it subjected to grow at the constant concentration of camel whey. Through the first three hours of incubation there were no significant differences between the growth of LAB strains obtained from cow and camel milks however, an evident differences were observed during the remaining 24 hours of incubation. After 6 hours of incubation LAB strains that obtained from camel milk grew increasingly whereas, the same species that obtained from cow milk could not able to progress and its optical density was almost constant. As represented in Figure (11) there is an apparent difference at the growth of LAB that obtained from the two sources. The optical density values of the bacterial growth after 24 hours of incubation established that LAB strains isolated from camel milk can grow optimally in the presence of camel whey at the same activity wherever it absence. In comparison with the behavior of same species of LAB that obtained from cow milk, it was noticed that cow milk LAB lost their activity and their growth declined sharply after 24 hours of incubation at the medium supplemented with camel whey.
As camel milk is rich in antibacterial proteins that control the growth of gram negative bacteria and peptidoglycan recognition protein which control the growth of gram positive bacteria and it could not detected in other Ruminants milk (Kappler et al., 2004), there is a need to examine the ability of LAB to resist the antibacterial effect of camel milk whey proteins.
According to Kappler et al. (2004) who detected that PGRP is found in camel milk only and the main role of this peptide is to control positive Gram bacteria and bound with peptidoglycan of the bacterial cell wall and thus inhibit LAB growth. Moreover, he reported that presence of exopolysaccharide (EPS) surroundings bacterial cell wall can avoid it from binding with PGRP. Based on these facts most of LAB isolated from camel milk should be EPS producers to save itself from the inhibition action of PGRP. This explanation is in agreement with Estiphano et al. (2016) who found that 85% of LAB isolated from camel milk had EPS production, and thus explain clearly the ability of LAB isolated from camel milk to resist its whey compared with those isolated from other sources such as cow milk.

Conclusion:-
This study showed new aspects about the behavior of LAB isolated from camel milk that can grow in complicated ecosystem like camel milk and it resists the camel whey that rich in antibacterial proteins. In addition it is recommended that that these species should be further studied according to selection criteria for dairy industries like EPS production, acidifying activities and stimulation of immunological system and adhesion the epithelium tissue to can benefit from their unique characters to produce functional dairy products.