Use of Pergularia tomentosa Plant Enzymatic Coagulant System in Fresh Cheese-Making

Introduction: Pergularia tomentosa is a saharan spontaneous plant belonging to Asclepiadacea family. It has several medicinal properties and uses in the human food as an auxiliary in cheese-making. The purpose of this study is the extraction of the coagulant enzymatic system (C.E.S) from the plant dried leaves and its use in the preparation of fresh cheese. Materials & Methods: The determination of the coagulant activity was carried out on a skimmed milk powder by Berridge’s method. Cow’s milk used for coagulant strength determination and cheese-making. Proteolytic activity. The chemical determinations of raw milk were carried out according to AFNOR analysis methods. A descriptive sensory evaluation was performed to determine the organoleptic properties of the produced cheese. The proteolytic activity was determined by measuring the aspartyl-protease units. Results: The main results of the phy-sico-chemical characterization show that its coagulant activity (R.U mL −1 ) was 97.19 ± 4.67, coagulant strength (SU) was 230.05 ± 0.37, proteolytic activity (μg·mL −1 ) was 0.33 ± 0.04 and coagulant activity/proteolytic activity ratio was 294.51. For the cheese made, it has presented the following results as % (g/100g): acidity 0.005, total dry extract 30.48 ± 1.490, fat 2.32 ± 0.02, fresh yield ± 0.09, dry yield 30.06 ± Moreover, sensory analysis results showed that fresh cheese prepared from Pergularia tomentosa dried leaves diluted enzymatic extract filtrate was characterized by whitish color, smooth tex-ture, with acceptable and satisfying lactic taste. Conclusion: The plant enzymatic system studied could be added to the list of plant rennet substitutes and, thus, contributes to the manufacture of enzymatic coagulation cheese.


Introduction
Saharan spontaneous plants are characterized by their particular mode of adaptation to the environment. Pergularia tomentosa, is a perennial shrub belonging to the Asclepiadaceae family [1]. This family plant represents an immense reservoir of potential compounds attributed to the primary and secondary metabolites. They have the advantage of participating through their biological activities thanks to their appropriate chemical structures in several industries of food, cosmetology, and pharmacology [2].
Pergularia tomentosa is a scandent or climber arising from a woody rot-stock, in dry savanna and wooded savanna. It is widely distributed across the Sahara desert, Mauritania and Mali to North Nigeria. It extends to the Congo basin and eastward across the Horn of Africa through Sinai (Egypt), southern Israel, Jordan and the Arabian Peninsula to the deserts of southern and eastern Iran, Afghanistan, Pakistan and India. Usually, it grows in sandy and clay soil areas of deserts [3] [4]. It has a lot of medicinal efficacy. Each part of the plant has specific medicinal properties. For example, roots are used for the treatment of pulmonary disorders, the whole plant against diarrhea, and dysentery. For the plant sap, it treats cutaneous and subcutaneous parasitosis [5].
In eastern Sudan peulh, herdsmen rub the latex onto the udder of cows to increase milk production. In semi-desert areas a little milk, in which stems have been soaked, is added to milk to curdle it for cheese-making. It is also reported to be given to goats as forage. The stems are reported to be occasionally used as famine food [6]. In Algeria, this plant commonly called in southern, "Ghalga" or "H'lib Edabba grows on the rubble, the edges of the paths and the degraded steppe courses. It is used as a hemostatic substance, fights against Leishmaniasis and relieves dental pain [7]. In cheese technology, coagulation is the key step in making cheese. Indeed, it consists of the transformation of milk into gel following physicochemical modifications which occur on the casein micelles. It is often the result of the action of enzymes of animal, plant or microbial origin. Rennet is the main coagulating enzyme used in cheese-making, but the increase in the consumption and production of cheese causes a shortage of rennet and very large fluctuations in its price [8].
The objective of this study is the extraction of the coagulant enzymatic system (C.E.S) from the Pergularia tomentosa plant leaves and its use in the preparation of fresh cheese. Physicochemical and sensory characteristics are determined on the obtained products. Thus, plant enzymatic system studied could be added to plant origin rennet substitutes list to diversify the milk coagulating agents list.
We remind that Calotropis procera plant of the same Asclepiadaceae Pergula-

Samples
1) Berridge's substrate: The determination of the coagulant activity was carried out on a skimmed milk powder of a medium heating quality (Molochansk Dairy; Ukraine) with moisture 3% and fat 0.15%. After its reconstitution at 12% (m/v) in CaCl 2 (0.01M) solution, the obtained milk, constituted the Berridge substrate. It was stored at 4˚C overnight to ensure the physicochemical balance, the stability and rehydration of micelles. To prevent microbial growth, 0.04% (w/v) sodium azide was added.
2) Cheese-making milk: Cow's milk used for cheese-making was provided from a farm located in Constantine city (Algeria). After milking, it was immediately transported to the laboratory in a cooler (4˚C).

Coagulating Enzymatic System (C.E.S.)
Pergularia tomentosa leaves were collected from Ain Diab region of Biskra city (south-eastern Algeria Figure 1). They were dried in the open air at room temperature and then powdered with a mortar. The powder obtained underwent aqueous maceration in sodium acetate buffer solution (pH = 5.5) for 2 hours at 4˚C, followed by filtration and, then, centrifugation at 4000 g for 15 minutes at 4˚C. The supernatant represents the leaves crude extract [10].

Physicochemical Composition
The chemical determinations of raw milk were carried out according to AFNOR analysis methods: dry extract, lactic acidity, pH, milk and cheese fats were determined according to (AFNOR) 1980 [11]. Total proteins were quantified by the Bradford method [12]

Sensory Analysis
A descriptive sensory evaluation was performed to determine the organoleptic properties of the produced cheese. A panel of thirty (30) subjects including fifteen (15) males rated the odor, texture, and flavor descriptors on a point scale from 1 to 15 [13].

Physicochemical Characterization of the Enzymatic Coagulant
System (E.C.S.) Ten (10) mL of Berridge's substrate (pH 6.6) contained in a test tube were maintained in a water bath at 30˚C. The timer was started when adding 1 mL of coa-
Five liters of raw cow's milk were filtered through gauze to remove large fragments and then heated to 60˚C for five minutes. The diluted enzymatic extract (1/100) of Pergularia tomentosa dried leaves (680 mL) was added with an increase in temperature to 75˚C. Then, the mixture was removed from the heat and was allowed to stand to favorise clotting. After firmness, of about 35 minutes, the curd was drained, then stored at 4˚C (Figure 2).   [18] was calculated on a wet and dry basis according to the following formulas: fresh curd quantity Fresh yield 100 milk quantity = × fresh curd quantity curd total dry extract Dry yield 100 milk quantity total milk dry extract

Physico-Chemical Characterization of Cow's Milk
Physicochemical characteristics of the raw milk used in the manufacture of fresh cheese are mentioned in Table 1.
The pH of raw milk used in cheese-making is 6 [21] of pH between 6.6 and 6.8. On the other hand, the titrable acidity is 17.51˚D, it is higher than that reported by Labioui (2009) [22] of 16.75˚D, and than of Rouissi (2018) [23] 15˚D. These results remain in conformity from 16 to 18˚D as declared by (AFNOR 1993) [24]. The average total dry extract content is 11.66%. It is lower than that given by FAO (1995) [25] of 12.8% and similar to that of Labioui (2009) [22] which is 11.75%. For fat, a value of 32 g/L is noted, slightly lower than cited by (AFNOR 1993) [24] for milk from 34 to 36 g/L and the value reported by Rouissi (2018) [23] of 42.6 g/L. However, it is close to 31.45 g/L, value announced by Labioui (2009) [22]. These differences 1-1 1-2

Milk filtration and heating
Coagulation by (C.ES.) extracted Cheese draining and molding Fresh cheese   [28]. Thus, the fresh milk used in the production of cheese meets the quality criteria related to fresh, non-acidic milk, with a dry extract and a fat content suitable for a satisfactory cheese yield.

Physico-Chemical Characteristics of Pergularia tomentosa Leaves Extract
Any coagulant enzyme is primarily characterized by its coagulant strength and proteolytic activities.  [29].
The protein content is greater than that of the enzymatic extract of Calotropris procera estimated at 3.74 mg/mL as reported by Aworh & Nakai (1986) [35] and also up than 26 mg/mL cardosine protein coagulant extract of Cynara cardunculus flowers [36]. For the proteolytic activity of 0.33 μg/mL, it is relatively lower than that given by Necib   climatic, soil and even leaf conditions (dry or fresh). In fact, each enzymatic extract has thermal and chemical stability and an adaptation with its catalytic medium linked to the appropriate reaction factors. Thus, these characteristics may be potential parameters for the Pergularia tomentosa enzyme use in the coagulation of milk and, therefore, in the manufacture of cheese.

Physico-Chemical Characteristics of Manufactured Fresh Cheese
The main physicochemical characteristics of fresh cheese are mentioned in Table 3.
The study results) show a pH of 6.38, relatively close to the pH 6.28 given by Benyahia-Krid et al., (2016) [9] for the "peulh" cheese type prepared from the coagulation of milk by the filtrate of Calotropis procera leaves. However, Necib & Tabet (2018) [33] give an average pH of 5.89 for fresh cheese prepared from Pergularia tomentosa latex.
This variation could be explained by the initial pH of the milk used and the coagulation method followed. Regarding the titratable acidity of 1.8 g/L, it is This difference in the total solids content could mainly be due to curd types obtained which are dependent on the initial milk composition, the type of coagulation as well as the type of drainage [14]. The fat content of 2.32 percent grams of cheese agrees with the value given by Codex Standard (283-1978) [37] which should be less than 20% for fresh cheese.
The average produced cheese (fat/dry) extract ratio of 7.61% confirms its belonging to the lean fresh cheese category according to the classification of Codex Standard (283-1978) [37], which stipulates a lower fat/dry extract ratio at 10%. The same source indicates that the unripened cheese of which the fresh cheese is ready for consumption shortly after its manufacture.
For the fresh cheese yield produced, it is 11.50% and 30.06% on a wet basis and on a dry basis, respectively. Necib & Tabet (2018) [33] present fresh cheese yields of 15.35% for cheese obtained by Pergularia tomentosa latex milk coagulation and 15.27% for that obtained by Pergularia tomentosa leaves filtrate milk coagulation. On the other hand, Benyahia-Krid (2016) [9], have presented a cheese yield on a fresh basis of 17.69% and on a dry basis of 55.45% for the Peulh type fresh cheese obtained by calotropaïn milk coagulation of Calotropis procera. For this same last plant and for the same Peulh type fresh cheese, Djeddi & Misky (2014) [29] noted a cheese yield of 7.69%, and Chella & Mamou (2018) [43] of 24.9%.
The yield depends closely on the type, quality of cheese produced, the milk composition used and the followed unit operations, mainly the draining method.
These aspects could be a result of the Pergularia tomentosa enzyme acts on milk caseins and minerals at high temperature over 58˚C, which confers a moderate compactness a firm (9.96 ± 1.71) and a crumbly (7.20 ± 0.88) structure.
On the other hand, and during cheese making, the heat clotting temperature might relatively mask the herbal leaves odor and taste. On the contrary, it might  . Sensory profile of fresh cheese from Pergularia tomentosa dried leaves extract.
develop lactic taste (8.00 ± 0.78) which is the main characteristic of fresh cheeses. Also, this cheese has an animal odour (7.93 ± 0.78) provided from bovine milk used for fresh cheese making. A slight non-unpleasant bitterness (2.2/15) (is) reported. This descriptor is intense (10/15) as reported by [33] for fresh cheese prepared from raw undiluted filtrate from Pergularia tomentosa. Several studies on the phytochemical composition of Pergularia tomentosa extract show its richness in polyphenols, saponins, amides, glucosides, tannins and alkaloids. These substances impart a bitter taste to products derived from this plant when they are found at a high concentration [44] [45] [46]. However, this bitterness seems to disappear-one of the main objectives of this study-by diluting the crude extract of Pergularia tomentosa and, consequently, reducing the concentrations of the substances responsible for the bitterness.
Although this bitterness is unpleasant to the taste, we would like to remember that bitter substances stimulate the function of liver and allow the body to food absorbed optimally process [47].
The sensory results obtained from the fresh cheese produced are similar and agree with those given by Benyahia-Krid et al., (2016) [9] for the Peulh type fresh cheese concerning its firmness, smooth appearance, dispersion and persistence in the mouth. This could be explained by the fact that the two plants Pergularia tomentosa and Calotropis procera are from the same Asclepiadaceae family and, therefore, have similarity in the properties and characteristics of their enzymes.

Conclusions
Fresh cheese made with the diluted enzymatic extract of Pergularia tomentosa plant has appreciable organoleptic characteristics for its whitish color, its smooth texture, as well as for its smell and its lactic taste. In addition, the disappearance of the relative bitterness in the mouth following the dilution of the leaves extract gave it a remarkable perception and an appreciable evaluation.  In fact, Pergularia tomentosa plant appears to be an interesting source of coagulant enzyme that could be added to the plant-derived rennet substitutes list. Thus, the physicochemical characteristics of the extract plant leaves reveal a significant coagulant potential which deserves to be exploited in the coagulation of milk and therefore in the manufacture of certain fresh or even ripened cheese.