Physicochemical profile and Lactic Acid Bacteria genera inhabit Egyptian raw camel , sheep , goat , buffalo and cow milks

Indigenous dairy products made of different milk sources are traditionally produced and consumed in a majority of African and Arabian countries. The aim of present study was to assess and compare physicochemical profile of camel, sheep, goat, buffalo and cow’s native milks in addition to isolation, identification and technological evaluation of their content of Lactic Acid Bacteria (LAB) genera to introduce a wider image that facilitate applications of these pasteurized milks along with isolated LAB strains in dairy industry development. Milks were examined chemically and via Fourier Transform Infrared (FTIR) spectroscopy. Sixty strains out of two hundred sixty-eight of LAB were selected based on assessment of their technological properties. Chemically, sheep and buffalo milks showed the highest content of protein, fat and ash. Goat and sheep milks possessed high content of lactose, which encouraged the inhabitance of Leuconostoc sp. to represented 48% and 18% respectively of their total LAB isolates. Some strains showed exceptional autolytic, photolytic and antimicrobial features (10, 15 and 12 strains respectively). Obtained findings when correctly applied would lead to develop an innovative dairy processing in Egypt. Key words– FTIR, Functional properties, LAB Isolation, Native row milks, Phenotypic identification; Proximate analysis. Introduction Milk as nature’s most complete food, considered daily source of nutrients in most countries. Studies on milk composition of dairy animals mainly concerned cow milk (85% of consumed milk), a lesser extent on goat and sheep milk, and rather rare studies on buffalo and camel in spite of their nutritional importance (Konuspayeva et al. 2009). Cow milk is the most universal raw material of manufactured dairy products (Dandare et al. 2014). Buffalo’s milk is ranked second in the world after cow’s milk (Ahmad et al. 2008). Camel milks play a major role and they are either home-consumed or sold (Yam et al. 2015). Sheep milk contains higher levels of total solids and major nutrient than other milks that affect coagulation time and rate, curd firmness, and amount of rennet used (Park et al. 2007). In recent years, with the huge issue in relation to green analytical technique, some scientist try to used environmentally friendly techniques. The FTIR technique has been gaining Microbial Biosystems 3(1): 12–24 (2018) ISSN 2357-0334 http://fungiofegypt.com/Journal/index.html Microbial Biosystems Copyright © 2018 Darwish et al. Online Edition


Introduction
Milk as nature's most complete food, considered daily source of nutrients in most countries.Studies on milk composition of dairy animals mainly concerned cow milk (85% of consumed milk), a lesser extent on goat and sheep milk, and rather rare studies on buffalo and camel in spite of their nutritional importance (Konuspayeva et al. 2009).Cow milk is the most universal raw material of manufactured dairy products (Dandare et al. 2014).Buffalo's milk is ranked second in the world after cow's milk (Ahmad et al. 2008).Camel milks play a major role and they are either home-consumed or sold (Yam et al. 2015).Sheep milk contains higher levels of total solids and major nutrient than other milks that affect coagulation time and rate, curd firmness, and amount of rennet used (Park et al. 2007).
In recent years, with the huge issue in relation to green analytical technique, some scientist try to used environmentally friendly techniques.The FTIR technique has been gaining interest for raw milk quality control, because of its high level of analytical capacity, low sample manipulation and use of fewer reagents, resulting in less time and lower costs (Fadzlillah et al. 2013;Coitinho et al. 2017).
Unfortunately, traditional cheese makers value the flavor that obtained by the use of raw milk, where the microbiota contributing to the ripening has a great influence on its specifications.Lactic acid bacteria (LAB) as starter and adjunct starter cultures, by producing acids and several lipolytic, proteolytic enzymes and antimicrobial agents, play an important role in preserving and producing flavor in cheese products that could encourage cheese makers to use pasteurized milk for lower incidence of food-borne diseases (Navidghasemizad et al. 2009).
The aim of this study was to provide a contribution concerning physicochemical profile of camel, sheep, goat, buffalo and cow Egyptian milks in addition to isolation, identification and technological evaluation of Lactic Acid Bacteria (LAB) genera inhabit these raw milks to introduce a wider image that facilitate applications of these pasteurized milks and isolated LAB strains in dairy industry development.

Sampling
Twenty five raw milk samples (5 samples per animal) were collected from different districts in Egypt, transferred under cooling conditions and kept refrigerated at 4 o C for analysis.

Physicochemical and nutritional characterization
All proximate analysis determined using the Official Methods of Analysis (AOAC International 2016).Moisture, ash, protein, fat and lactose contents were determined by oven drying, furnace, Kjeldah, Gerber and titrimetric methods respectively.pH value was determined using pH meter (AD1030 ADWA, Romania).Titratable acidity of the samples was expressed as the percentage equivalent lactic acid according to (Ling 1945).Energy was calculated (Calories/ 100mL) according to the following equation: (Protein*4+ Carbs (Lactose)*4+ Fat*9) (WHO/FAO 2002).

Fourier transform infrared (FTIR) spectroscopy
For wider image of milks' characteristics, FTIR spectra of examined milks were studied to mark functional groups using Fourier transform infrared spectrophotometer (Shimadzu FTIR-8400 S, Japan) equipped with (ATR 8000A).The range of spectrophotometer was from 4000-400 cm −1 (Cerqueira et al. 2011).

Isolation and phenotypic identification of LAB strains
For enrichment, one mL of milk samples were incubated in 10 mL sterilized reconstituted skim milk (RSM) (12.5%), at different incubation temperatures; 30 o C, 42 o C and 37 o C for 24 h.Selective media M17 and MRS (Biolife, Italy), were used for the isolation of LAB aerobic strains (De Man et al. 1960).The isolates were purified using streak plate method.Colonies were picked up according to shape and color, Gram-positive, catalase-negative isolates were phenotypically identified to the genus level using (CO 2 production, growth at 45 o C, 10 o C, growth in of 6.5% NaCl, in pH 9.6 and in SF medium) and biochemical characterization via carbohydrate fermentation (MacFaddin 1976).The identified isolated strains were stored at -20 o C in RSM (12.5%) supplemented with 15% glycerol and were registered in Faculty of Agriculture Saba Basha, Alexandria University Culture Collection (FABA).

Technological characterization of selected identified strains
Based on earlier observations, that suggested a causal relationship between microbial taxa and flavor, flavor development in milk cultures is one of the most important attributes (Walsh et al. 2016).Depending on a preliminary experiment of sensory evaluation of pre-grown cultures according to Ayad et al. (2004), sixty isolated LAB strains were selected for technological characterization; 24 isolates from camel milk, 11 from sheep milk, 6 from goat milk, 13 from buffalo milk and 6 from cow milk.The autolytic activity was determined as the percentage decreased in the absorbance (OD 650 ) at time intervals comparing the strain growth to blank M17 broth (Allam et al. 2017).Proteolytic activity represents the strain ability to hydrolyse milk protein as halo of proteolysis around the strain growth (Ayad 2001).Antibacterial activity was determined by agar well-diffusion assay against E. coli obtained from Netherlands Institute for Dairy Research (NIZO) according to Ayad et al. (2002).Exopolysaccharides production was determined by touching incubated strains with a sterile inoculation loop, strains were considered positively EPS producer if the length of slime exceeded 1.5 mm according to Knoshaug et al. (2000).

Statistical analysis
Statistical analysis was performed using Analytical Software SPSS® 13.0 (Statistical Package for the Social Sciences).Differences were considered significant at p < 0.05.All experiments were performed in five replicas.

Chemical and nutritional characterization of raw milks
Chemical and nutritional characteristics of the five raw milks; camel, sheep, goat, buffalo and cow are illustrated in Table (1).Specific gravity (SG) values ranged between a minimum of 1.033 (in cow milk) and a maximum of 1.036 (in sheep milk).Normal milks were reported to record specific gravity values not less than 1.030 (LR 30) to guarantee absence of adulteration by water addition.Accordingly, obtained results correlated with fat, TS and SNF of analyzed milk samples.The highest SG showed by sheep and buffalo milks (1.036 and 1.035) accompanied with their high TS, SNF and fat content (21.47% and 17.36%, 12.22% and 10.99%, 9.25% and 6.64% respectively), whilst, the lowest SG of cow milk (1.033) reflected lower content of TS, SNF and fat (13.13%, 9.92% and 3.21% respectively).On the other hand, the equal SG values of camel and goat milks (1.034) indicated the insignificant differences in their TS, SNF and fat content (14.45% and 14.80%, 10.03% and 10.55%, 4.43% and 4.25% respectively).Anyways, the fat content is used to be a measure of satisfaction indicating the overall milk quality taking in account TS content to avoid suspicion of adulteration using other fats (Kanwal et al. 2004).
Table (1) results reflected the energy as nutritional parameter (calories gained by 100g of milk sample), which arranged the milks as follows; sheep, buffalo, goat, camel and cow milks with energy values of 121.67, 94.24, 77.55, 70.97 and 59.39 calories respectively.Noticing that goat milk exceeded camel milk in energy which could be attributed to its high lactose content (5.73%), the principle carbohydrate in milk, that exceeded significantly the other tested milks followed by sheep milk lactose content (5.56%), while in the three other milks lactose content was around 4%. Ash content results reflected the valuable minerals content of camel and sheep milks (0.81% and 0.77%) followed by buffalo, cow and goat mineral content (0.70, 0.66 and 0.59% respectively).
pH values were near the neutral pH which ranged between 6.60 in camel and cow milk and 6.88 in sheep milk, and subsequently reflected on titratable acidity values which ranged between 0.133% in sheep milk to 0.155% in cow milk.
Results of camel milk surpassed North African camel milk values reported by Konuspayeva et al. (2009) except for lactose content that was 4.65%.Park et al. (2007) reported comparable SG and SNF results of sheep milk in present study, while differed in parameters of cow milk.Obtained analysis of goat milk agreed with Da Costa et al. (2014), except for protein results that scored higher content in current samples, and were near to results of Greece and Spain breads reported by Raynal-Ljutovac et al. (2008), which could be relied to similar weather condition in Egypt and the two countries.Buffalo characteristics matched with The Nili-Ravi Chinese buffalo breed reported by Han et al. (2007).However, complexity that underlies regional differences, including breeds, feeding conditions and seasonal or physiological variations, comprises a difficulty in comparing milk physical and chemical characteristics with previous results (Konuspayeva et al. 2009).

Fourier transform infrared (FTIR) spectroscopy
Fourier Transform Infrared (FTIR) spectroscopy is a qualitative rapid technique for the determination of the "fingerprint" of organic compounds because their functional groups exhibit characteristic vibrational absorption/ transmittance frequencies in specific infrared region (Durazzo et al. 2015).Figure (1) demonstrated FTIR results of the five milk types from different sources, camel, sheep, goat, buffalo and cow.
The milk protein connected bands are shown in the transmission bands observed at 3433, 3478, 3428 and 3426 cm -1 in camel, goat, buffalo and cow milk respectively (Nicolaou et al. 2010) and at 1540 to 1650 cm -1 which attributed to amide due to C=O and amine groups (N-H) (Durazzo et al. 2015).Obtained results could not reflect the minor significant differences exhibited in milks' protein content (Table 1).At the same time, the typical transmittance spectra for water located between 3650-3000 cm -1 represented in the hydroxyl group (O-H) (Coitinho et al. 2017).The fat relating bands were clearly illustrated at the transmissions observed in stretching bands presented in all milk samples between 2924 and 2855 cm -1 corresponded to the C-H of methyl and methylene groups located between 2900 and 2800 cm -1 according to Koca et al. (2015).Free fatty acids (FFA) reported to be present in milks resulted in carbon chains, which were most obvious in the symmetric C-H stretching region.Further, the region represented in milks between 1740 to 1520 cm -1 is typically used to detect the presence of carboxylic acid groups.This was an indication of presence of short chain fatty acids (SCFA) such as acetic, butyric and propionic acids which reported to be present in the milk of ruminant animals (Bourassa et al. 2016).This observation in accordance with what was previously reported by Nicolaou et al. (2010).
Lactose, milk sugar is a disaccharide composed of the monosaccharides D-glucose and D-galactose, joined in a ß-1,4-glycosidic linkage.The chemical name for lactose is 4-0-ß-Dgalactopyranosyl-D-glucopyranose.The bands at 1429 to 1447 cm -1 , represent the bending vibration of C=O and C-O-C present in the pyranose ring.The bands between 1163 and 1011 cm -1 is assigned to C-O-C stretching of 1→4 glycosidic bond ring vibration and C-OH bending and is recognized as the characteristic of polysaccharide compounds.Furthermore, the band at 702 to 714 cm -1 , represents glycosides linkages attributable to glucopyranose (Kong et al. 2007).Moreover, beside saccharides, the area 900 to 1680 cm -1 was reported to include frequencies of bioactive molecules amides (Durazzo et al. 2015).It is noteworthy that bioactive peptides derived from all milk types proteins are of great interest due to their diversity and health benefits (El-Salam et al. 2013).In summary, the presented FTIR features of camel, sheep, goat, buffalo and cow milks made a clear demonstration of all milk types composition that confirm the earlier conclusion of Coitinho et al. (2017).

Isolation and phenotypic identification of LAB strains
Two hundred sixty-eight active strains were isolated and phenotypically identified from all milk sources under investigation.Biodiversity of wild LAB inhabit Egyptian raw milks distributed as percentage of phenotypic cvriteria was presented in (Figs.2-6).Above figure (Fig. 2), showed that the 64 isolates were distributed as follows; Enterococci (43%) and Lactobacilli (36%) representing the predominant genera, with low contribution of Streptococcus thermophilus and Lactococcus (13% and 8% respectively).Similar observation was recorded by Fguiri et al. (2015) when ten selected isolates from camel milk were identified as Enterococcus faecium assuring its dominance.On the other hand, Abbas et al. (2014) succeeded to isolate Lactobacilli strains from different groups from camel milk.Five genera were recovered from goat milk samples represented by 35 isolates namely: Enterococcus, Lactococcus, Leuconostoc, Streptococcus and Lactobacillus (Fig. 4).Leuconostoc sp.represented almost 48% of LAB isolates assuring this genus dominance (Fig. 4).The metabolism of citrate which produces flavor compounds such as diacetyl and acetoin is an important pathway of the Leuconostoc species (Schmitt et al. 1991).Addition of lactose to Leuconostoc cells was reported to increase the growth rate (Huang et al. 1994), this observation could be connected to milk chemical composition (Table 1) where goat milk showed the highest lactose content amongst the other milks (5.73%) followed by sheep milk (5.56%) which embraced Leuconostoc 48% and 18% of their LAB isolates respectively.The distribution of 76 strains isolated from buffalo milk showed that Enterococci representing by 46% of total isolates (Fig. 5).Lactobacilli represented 36% of the total isolates split to majority of Group A (21%) and almost equal percentages of Group B and C (8 and 7% respectively).Relevant work of Rizqiati and hid coworkers (2016) supported our obtained results by isolating Lactobacilli strains as a dominant taxa from Indonesian buffalo milk.
LAB strains isolated from cow milk were 55 and their biodiversity represented in figure (6).Lactobacilli strains dominated the other genera with a percentage of 43% harbored a majority of Group B (27%) and low percentages of Group C and Group A (9 and 7% respectivly).Enterococci sp.represented 38% of isolates, while Lactococci showed 17% and fainted representation of St. thermophilus strain (2%).These results in agreement of the study carried by Abdullah et al. (2010).

Technological characterization of selected strains
Technological characteristics of 60 selected strains belonging to 16 LAB genera isolated from the 5 examined native milks are summarized in Table (2).Results revealed that only 10 strains showed autolytic activity.The majority of autolytic strains belonged to camel milk (6 strains), three E. faecium and one strain of E. faecalis, E. pseudoavium and L. lactis subsp.lactis.Three strains of cow milk selected isolates showed autolytic activity, L. lactis subsp.cremoris, L. lactis subsp.lactis and Lb.plantarum.Only one strain isolated from buffalo milk belongs to E. faecalis.It is noticeable that more than 50% of autolytic strains belong to Enterococci sp., which reported to play an important role in the production of various traditional fermented food products (Allam et al. 2016).
Fifteen strains recorded proteolytic activity.Six strains originated from cow milk, E. durans, L. lactis subsp.cremoris, L. lactis subsp.lactis, Lb. plantarum and two strains of Lb. paracasei subsp.tolerans.Sheep milk proteolytic strains belonged to Enterococci, two E. faecalis, two E. faecium and one E. durans.Only two strains of each of camel (L.lactis subsp.lactis and Lb.rhamnosus) and buffalo milk (E.faecalis and Lb.plantarum) showed proteolytic activity.Despite of wild LAB stability in milk and cheese, they harbor active amino acid convertases, which is interesting for flavor formation in the manufacture of fermented dairy products (Allam et al. 2017).a Strains considered autolytic when score represents 30% or above.b Fast, medium and slow; when a Δ pH of 0.4 unit was achieved after 3, 3-5 and >5 h respectively.
Buffalo milk selected strains showed antimicrobial potentiality surpassed other isolates when owned eight out of twelve strains with antimicrobial activity which distributed as follows, three E. faecalis strains, one strain belong to E. durans, E. seriolicida and three Lactobacilli strains, Lb. plantarum, Lb. casei and Lb.paracasei subsp.paracasei.The other four strains were Leu.oenos from sheep milk, E. faecalis and E. durans from goat milk and Lb.paracasei subsp.tolerans from cow milk.These results can support De Martinis et al. (2016) who suggested potential applications of buffalo milk LAB isolates with proved proteolytic activity and production of antimicrobials in dairy production.
Most selected strains (65%) showed slow acidification rate, while medium and fast acidifiers represented 21.7% and 13.3% respectively.All eight fast acidification strains belonged to Enterococci sp. and Lactobacilli sp.isolated from camel milk.On contrary, all goat and cow LAB isolates showed slow acidification rate.Allam et al. (2017) reported that slow acid producing strains could be applied to soft cheese and other dairy preparation that fast acidity is not considered a cornerstone in their manufacture.None of selected strains found to produce EPS.

Conclusion
Present work emphasized the differences in compositions of camel, sheep, goat, buffalo and cow Egyptian milks that could be a guide for dairy manufacturers.Sheep and buffalo milks showed the highest concentrations of protein, fat and ash in comparison with other studied milks, which should be taken into account affecting curd firmness, coagulation time and rate and amount of rennet used.FTIR results supported chemical analysis performing protein, fat, lactose and bioactive peptides.High lactose content revealed by goat and sheep milks suggested to be reflected on their LAB biota, which encouraged the inhabitance of Leuconostoc sp. to represented 48% and 18% respectively of their total LAB isolates.Furthermore, some isolated strains showed exceptional features such as, Lb. plantarum from buffalo milk and Lb.paracasei subsp.tolerans from cow milk that brought together photolytic and antimicrobial potentials, while L. lactis subsp.lactis from camel milk, L. lactis subsp.cremoris, L. lactis subsp.lactis and Lb.paracasei subsp.tolerans from cow milk that assembled autolytic and photolytic activities.These findings and LAB strains when correctly employed in dairy processing as starter or adjunct cultures, this would lead to developed innovative dairy products.

Fig. 2 -
Fig. 2-Percentage distribution of 64 strains of LAB isolated from camel milk.

Fig. 3 -
Fig. 3-Percentage distribution of 38 strains of LAB isolated from sheep milk.

Figuer ( 3
Figuer (3) expressed the distribution of the 38 isolates of LAB recovered from sheep milk.Lactococcus sp.came first by recording 42% followed by Enterococcus sp.(32%) out of all recovered taxa.Other taxa namely Leuconostoc sp. and St. thermophilus recorded lower percent as 18% and 8% respectively with the complete absence of Lactobacilli sp.These results in contradictory with results obtained byIranmanesh et al. (2012) who succeeded to isolate Lactobacilli strains from Iranian Ewe milk.

Fig. 4 -
Fig. 4-Percentage distribution of 25 strains of LAB isolated from goat milk.

Fig. 5 -
Fig. 5-Percentage distribution of 76 strains of LAB isolated from buffalo milk.

Fig. 6 -
Fig. 6-Percentage distribution of 55 strains of LAB isolated from cow milk.

Table 1
Physicochemical and nutritional characteristics of 100 mL raw milks.Data were presented as the mean of five replicates followed by the standard deviation (mean ± SD). ,c,.. Means values in the same row marked with unlike letters are significantly different (p<0.05)

Table 2
Technological characteristics of selected LAB strains isolated from raw milks