Phytochemical screening of J. phoenicea L. leaves
The qualitative phytochemical screening results of
J. phoenicea L leaves were given in Table 1. According to the table below, the phytochemical screening showed the presence of several compounds namely coumarins, tannins, terpenoids, free quinones, alkaloids salts, flavonoids, starch and also many others, with the absence of alkaloids bases, emodin, saponins and reducing sugars.
Monitoring the fermentation of raw goat milk with J. phoenicea L. leaves
Detection of antibiotic residues
The wells diffusion method results indicate an absence of inhibitory zone around the wells and consequently inhibitory residue-free, namely antibiotic one. This confirms the hygienic quality of the analyzed sample that meets national regulations (JORA n.39, 2017). Also, the susceptibility assay confirms these results, where the reference bacterial strain
E. coli was susceptible to trimethoprim/sulfamethoxazole and tetracycline, while
S. aureus strain was susceptible to oxacillin, erythromycin, cotrimoxazole and tetracycline (Table 2).
The wells’ diffusion method carried out for the detection of antibiotic residues confirms the hygienic quality of the analyzed sample which meets national regulations (JORA n.39, 2017). Milk without residues of antibiotics would increase consumer safety and competitiveness at the level of national and international trade According to
Roostita et al., (2011), even at low concentrations, the consumed antibiotic residues can cause health problems such as allergies, intoxication and antibiotic resistance. Moreover, milk containing antibiotic residues cannot be treated with a microorganism starter. Thus, a study conducted by
Rahimah et al., (2019), showed that
E. coli,
S. aureus and
Salmonella sp strains isolated from raw milk in Indonesia were resistant to chloramphenicol, trimethoprim and ampicillin, due to the overuse of antibiotics in the livestock industry which has led to a high diversity of multiple antibiotic resistance genes.
Physicochemical parameters
Fig 3 shows the change in pH, temperature and titratable acidity of goat milk (the test and control samples) during fermentation.
We noticed an increase in the titratable acidity of the control and the test samples, at the beginning of the 2
nd day with higher values for the test sample compared to the control one. However, the pH parameter experienced a decrease over time,
i.e. an increase in titratable acidity. The mean temperature of the test and control samples was 17,86±1,18°C and 17,98±0,91°C, respectively. This variation depends on the ambient temperature of the milk as an extrinsic factor.
The pH is a parameter that determines the storability of foods. The pH measurement provides precise information on the state of the freshness of milk (
Luquet, 1985). It is one of the main obstacles that microbial flora must overcome to ensure its proliferation. However, both parameters: the pH and the titratable acidity of the milk depending on the casein and on the mineral salts’ content, extrinsic conditions such as temperature, endogenous flora or contamination load, biochemical components such as lactose content that depends not only on the breed but also on the stage of lactation and the hydration status of the animal. Also, the level of compliance with hygiene practices during the milk handling process (milking, transport, processing and storage)
(Tantaoui et al., 1983; Boubekri, 1984;
Labioui et al., 2009; Benyagoub et al., 2013; Benyagoub and Ayat, 2015).
The decrease in pH values can be explained by the biodegradation of the lactose into lactic acid for the control sample, as well as the effect of juniper leaves added to the test sample where the
J. phoenicea L. aqueous leaves macerate had a pH value of 5,29, which can provide real protection against deterioration caused by unwanted microorganisms (
Leksir and Chemmam, 2015).
The temperature as an extrinsic factor has an impact on chemical reactions, enzymatic activity and the rate of microorganisms’ growth in food. According to the results of the analyzed samples’ temperature, little variation was recorded, where the ambient temperature was relatively stable during the period of analysis.
However, the susceptibility assay results showed the absence of antibiotic residues in the analyzed sample where their presence will affect the process of the natural fermentation of milk (
Melahi and Benhila, 2017). Moreover, well progress of the fermentation process was confirmed by the evolution of both physicochemical and microbial parameters of the milk over time.
Bacterial parameters
Total aerobic mesophilic flora
Fig 4 shows the evolution of the total aerobic mesophilic flora of goat milk (test and control samples) during fermentation over time.
The total aerobic mesophilic flora is a good indicator of the hygienic quality of milk, stability of products as well as the quality of facilities (
Guiraud, 2003). Its presence in high load is justified by non-compliance with hygienic practices during milking, transport, processing, or storage. Fig 4 shows an increase in the bacterial load of the TAMF incubated at 22 and 30°C ranging from 3.2 to 7 Log
10 CFU/mL, which was important for the control sample compared to sample one. Noting that the bacteriological quality of the collected milk complies with national regulations.
The TAMF’s load of the control sample was higher compared to the test sample. These results can be explained by the effect of phytochemical compounds of juniper added.
Contaminants and pathogenic bacteria
Coliforms
Fig 5 shows the evolution of coliform organisms (total coliforms) and thermotolerant coliforms (fecal coliforms) of goat milk (test and control samples) during fermentation.
The presence of fecal coliforms is considered as an indicator of strong fecal contamination of animals (case of mastitis) or human origin due to lack of hygienic practices during milking or poorly cleaning and sanitizing the utensils used for milk collection (
Boufeldja, 2017).
The thermotolerant coliforms load in the collected milk complies with national regulations, but over time, the bacterial load increases to 3,85 Log
10 CFU/mL for the control sample. However, the test sample experienced a decrease in the microbial load going from 3,23 to 2,3 Log
10 CFU/mL.
Pathogenic bacterial strains
Fig 6 shows the evolution of contaminating and pathogenic bacteria, namely coagulase-negative Staphylococci,
Salmonella spp and
Listeria monocytogenes in goat milk (test and control samples) during fermentation.
At the reception, the sample had a load of coagulase-negative Staphylococci of 1,8 Log
10 CFU mL. However, national regulations set a load of m= 2,47 Log
10 CFU/mL and M= 3,47 Log
10 CFU/mL for coagulase-positive Staphylococci (CoPS).
The control sample showed an increase in the staphylococcal load up to a maximum value of 2,4 Log
10 CFU/mL which was still within the range given by national regulations. After 2 days of storage, the test sample revealed a maximum load of 2,3 Log
10 CFU/mL, then a decrease in this bacterial load was noted. However, no contamination was revealed by the pathogenic strains
Salmonella spp and
Listeria monocytogenes.
The contamination of milk by pathogenic microorganisms namely
Staphylococcus spp,
Salmonella spp and
Listeria spp is a possible sign of one (
Rainard and Poutrel, 1993;
Feknous et al., 2018; Benyagoub, 2018;
Benyagoub, 2019):
- Direct contact of the udders with dirty soil (wounds, unwashed udders before milking).
-Non-compliance with hygienic practices during milking at the farm level.
- Mastitis infection.
- The person who practices milking may be the carrier of a disease that can contaminate the milk (oronasal sphere, skin, wounds).
Thus, to produce a good quality and safe to consume milk, good handling and management are needed from the beginning of milk production to reduce the risk of microbial contamination and growth in milk
(Rahimah et al., 2019). However, the absence of pathogens in both samples, as well as the decrease in the coliform load in the test sample, can be justified by the good health of the animal and by the action of the acidity of the product and the effect of
J. phoenicea L. leaves phytoconstituents on the growth of contaminating and pathogenic microorganisms, where the major phytoconstituents as secondary metabolites were similar to the results reported by
Draoui et al., (2020).
Knowing that the antibacterial action of
J. phoenicea L. leaves varies from one microorganism to another because each bacterium has its characteristic. Several studies confirm the antiseptic action of
J. phoenicea species, namely
Aljaiyash et al., (2016); Draoui et al., (2020).
Concerning its use and benefit, the twigs, leaves and fruits of Phenician juniper are used in traditional medicine and their phytochemical compounds are incorporated in pharmaceutical preparations particularly for antiseptic use, attributed to the presence of essential oils.
The leaves are used as a herbal decoction against diabetes, diarrhea and rheumatism, while the dried and powdered fruit can cure skin ulcers and abscesses
(Mansouri et al., 2011). The leafy branches are used for the production of plant tar to treat some cases of eczema and inhalation for asthma, bronchitis, headaches, dizziness and to control arthritis (
Abdelli, 2017).
Lactic acid bacteria
Fig 7 shows the evolution of lactic acid bacteria of goat milk (test and control) during fermentation.
An increase in the lactic acid bacteria load for the control sample was revealed from 4,6 and 3 log
10 CFU/mL to 6,3 and 5,84 log
10 CFU/mL for the lactobacilli and Streptococci groups, respectively. While the test sample experienced an increase in the streptococci lactic flora load after reporting a decrease of the lactobacilli load so that at the end of the fermentation it was 5.08 and 4.95 log
10 CFU/mL, respectively.
The evolution of pH and titratable acidity results in an increase in the microbial load that makes up the indigenous microflora of milk. During fermentation, the transformation of lactose into lactic acid creates a favorable acid environment for the growth of endogenous milk flora.
Fermented milk revealed an increased load in lactic acid flora. Through the proteolytic activity, lactobacilli species attack the casein which releases the peptides allowing streptococci to continue their growth. Moreover, streptococci stimulate lactobacilli by producing formic acid. Thus, the increase in lactic streptococci comes from the reduction of lactobacillus loads which were responsible for the acidity of the product.
The lactic acid flora loads in the test sample were lower compared to the control one. This was probably due to the high acidity of the sample supplemented with juniper leaves which has a selective effect on microbial growth.
Sensory properties
The results of the sensory analysis were presented in Fig 8.
According to the graphical representation of the sensory analysis, the tasters focused on the two offered products, where the descriptors that experienced a difference were mainly as follows:
The tasting intensity for the acidic taste character, the odor intensity for the two characters (the herbaceous and sour smell) and the viscosity of the product, which produced a rating difference of appreciation where the control sample (B) was the most preferred one compared to the test sample (A).
The descriptive sensory analysis allowed us to describe the characteristics of the samples which according to the tasters:
• The color of the test sample was different from the control sample, due to the addition of the juniper leaves, which changed the color into dull white.
• In terms of smell, the tasters judged that the control sample was characterized by an acidic odor; though the test sample was more acidic, the addition of aromatic juniper leaves gave a herbaceous odor which hides the acidic smell of the sample.
• The control sample had high viscosity compared to the test sample (medium viscosity) probably because of the high lactic acid flora load, revealed through microbiological analysis, which produces more exopolysaccharides leading to an increase in the viscosity and improving the texture of fermented milk (
Douaer, 2018).
• The taste, the aftertaste and the general appreciation: The tasters judged that the taste of the test sample was acidic, where this character was not only attributed to the fermentation process, but also to the acidity provided by adding juniper leaves, which left a strong aftertaste in the mouth. According to the tasters, it was probably due to the relatively high m/v ratio (the weight of the added juniper leaves compared to the volume of the used milk). While the strong acid/milky taste of the control sample comes from the transformation of lactose into lactic acid and the natural milky taste allows it to have the best score of 5/5 on the established scale of preference which was very pleasant.
In addition to the concentration of aromatic juniper leaves, lactic acid bacteria also release flavor compounds and participate in the development of some organoleptic characteristics relating to the aroma and milky taste.