| Livestock Research for Rural Development 30 (4) 2018 | Guide for preparation of papers | LRRD Newsletter | Citation of this paper |
Recently, enzymes from plants and microorganism are using as a milk clotting alternatives in cheese production industries. The potential milk clotting characteristics (milk-clotting activity (MCA), syneresis capacity (SC) and speed and cheese yield (CY)) of the three species of plants extracts - Solanum incanum, Ficus carica and Rhus natalensis -compared with rennet for cheese making. The study was done in the dairy laboratory of Wukro St.Mary College, Tigray, Ethiopia from August to October 2015. The Experimental treatments were replicated four times using completely randomized design. The highest MCA indicated in milk clotting time (min) obtained using F. carica crude latex. The average SCs produced by calf rennet and S. incanum juice were similar while that of R. natalensis extract showed higher SC. F. carica latex showed relatively lowest SC and this may be due to high water retaining property in the coagulum. The use of F. carica latex as a coagulant gave the highest curd yield than chymosin, the extracts of S. incanum and R. natalensis. The relatively lowest fresh cheese yield observed by application of R. natalensis extract may be attributed to very high proteolytic property of the plant, indicating that it is relatively inconvenient in economic terms. The clotting activity of all the three plant extracts was significantly associated with concentration of the coagulants. The overall clotting characteristics (MCA, SC and CY) from F. carica appear to be more suitable as a milk clotting plant for cheese making.
Key words: cheese making, Ficus carica, milk clotting plants, Rhus natalensis, Solanum incanum
The commercial bovine rennet enzyme used to coagulate milk in cheese making is too costly (Ahmed et al 2010); and the need of calf sacrifice makes it difficult to use it at commercial as well as small-scale dairy production levels in Ethiopia. Moreover, the global shortage of this enzyme together with the increasing demand for cheese production in the world is imposing researchers to explore alternative milk clotting means from plants (Bornaz et al 2010). Several authors (Guinee and Wilkinson 1992; Broome and Limsowtin 1998; Fox 2000) have reviewed that proteases from microbial and plant sources can substitute calf rennet (Chymosin) to coagulate milk. For instance, in Mediterranean and near Mediterranean countries, in some South America countries (Chile and Argentina) (Campos et al 1990) and Africa (Guiama 2010), the use of plant-originated enzymes as milk coagulants was well recognized. The flowers of artichoke (Cynara scolymus), latex from the fig tree (Ficus carica), Calotropis procera, Solanum dubium and Euphorbia nivulia are among the well-studied ones ( Nouani et al2009;Bornazet al 2010; Alirezaeiet al 2011; Badgujar and Mahajan 2012). Fresh kiwi fruit ( Actinidia deliciosa) was also proven as potential coagulant for cheese production (Mazorra-Manzano et al 2013).
In agro-pastoral community of Ethiopia, latex from fig tree ( Ficus carica) refereed locally as ‘Beles’, also traditionally used to coagulate shoats’ milk mainly by goat shepherds in rural areas (personal observation and discussion with localities). Similarly, juices from Solanum incanum berries and Rhus natalensis leaves also observed to be used by rural farmers. However, the commercial farms in Ethiopia haven't exploited these plants due to absence of researches that attempted to characterize and standardize the use of these plants for milk clotting purpose.
Enhancing cheese production using locally available plant proteases can be one of the approaches for processing dairy products and the overall development of dairy production in Ethiopia. Studying the milk characteristic of these plants is of great importance to approve the fully utilization of the plants. Therefore, we designed the present study to determine the milk-clotting characteristic of the most commonly used plant - Ficus carica, Solanum incanum and Rhus natalensis extracts, compared with calf rennet and characterize the effect of concentration of these proteases on clotting activity in cheese making.
The agro-pastoral communities in northeastern part of Ethiopia (Irob in Tigray and Ab‘alá in Afar), traditionally use a number of plants for milk clotting. Among these, the three most commonly used ones - Ficus carica, Solanum incanum and Rhus natalensis were chosen for laboratory experiment. The experiment was done at Dairy laboratory of Wukro St. Mary College, 40km north of Mekelle, the capital of Tigray region, northern Ethiopia. The town is found at 2140-2250m above sea levels with a climate of Weina Degua. The total size of the town is 860 hectares, with comfortable environment for residential, has an average temperature of 19.730c and annual average rainfall of 690.25mm. It has three administrative kebelles, namely, Hayelom, Agazi and Dedebit with population size of 18971, 6915 and 5852, respectively (OFED 2008).
Crude enzyme of Ficus carica in the vicinity of the St. Mary’s College dairy laboratory was collected early in the morning by systematic incision of succulent stem and fruit of a green healthy plant. The milky latex was drained into a clean test tube (Badgujar and Mahajan 2012). Fresh juice from the berries of Solanum incanum simply cut in to parties and squeezed by a Juicer in to a beaker. The extracts from fresh Rhus natalesnsis leaves grounded and filtrated through cheesecloth by centrifugation at 12000 ×g for 20 min. The latex, juices and extracts from fromFicus carica, Solanum incanum and Rhus natalesnsis respectively were kept immediately after preparations in a refrigerator at +4 οC in the laboratory until used (for about 2 hours). Calf rennet (Natural liquid rennet - 90 cc, Nievi, Spain), obtained from Wukro St. Mary College was used as a control.
Mixed milk samples from eight hybrid Zebu (Bos indicus) × Holstein Friesian (Bos taurus) cows (>75% Friesian) were collected from the Dairy farm of Mekelle University, Department of Animal, Range land and Wildlife Sciences, Tigray, Ethiopia. Before conducting the experiment, raw milk composition and milk acidity was determined, using Lactoscan Milk Analyzer (Model S60, MB VERS 560, Serial No. 2995, Bulgaria). The average chemical composition of milk used for cheese making had 3.11% protein, 3.6% fat, 12.17% total solids, 0.72% ash and 6.6% pH. The milk was stored at +4 οC in refrigerator, until used, roughly for three hours. Before addition of extracts, the initial milk pH of 6.6 adjusted to 5.6 by adding lemon juice, and heated to 32 oC to facilitate clotting and treated.
The experiment consisted of four treatments, each with three replications using a completely randomized design. The three treatments comprised of latex extracts from succulent bark part ofFicus carica (T1), juices of Solanumincanum berries (T2), and extracts of Rhus natalensis leaves (T3). Calf rennet was used as control (T4).
Milk clotting activity (MCA) of extracts expressed in terms of time (minutes) required to induce clotting (coagulation). Less milk clotting time (MCT) indicates higher MCA and vice versa (Berridge 1952). The juice of Solanum incanum and Rhus natalensis extracts and crude Ficus carica latex added at a proportion of 0.1 ml per 10 ml of milk. Rennet used at a dose of 0.3 ml/l of milk. The first onset of clotting continuously examined. The clotting time in minute was recorded when discrete particles were visible.
The cheese was produced according to the procedure described by O’Connor (1993) with slight modifications. Briefly, the milk heated to 75 o C and allowed it to cool to 35o C, rennet at a dose of 0.3 ml/l and plant coagulants at 0.1 ml per 10 ml of milk added to the milk and stirred for about three minutes. Then the milk was let for two hours and separated curd from whey by using Sieve. Cheese yield (g/l) was expressed as the ratio of curd mass obtained after 4 hour of drainage to the volume of milk used.
Syneresis, according to Scherer (1989), is defined as spontaneous contraction of a gel, accompanied by expulsion of liquid from the pores. Syneresis capacity and speed of whey expulsion was determined according to Bornaz et al (2010). Whey separated from the curds by centrifugation at 4000 × g for 10 min without cutting the curds after 90 min of clotting. Syneresis capacity of curds produced by all coagulants calculated as the ratio of the mass of whey obtained to the mass of milk used. The speed of whey removal estimated after curds obtained after one hour and cut into small cubes and put into perforated cheese molds. The mass of the whey measured at 15 min time interval for the first one-hour and every 30 min subsequently up to 4.5 h.
Effect of concentration of Ficus carica, Solanum incanumand Rhus natalensis crude proteases on milk clotting activity studied by adding different volume - 0.1 ml, 0.2 ml, 0.3 ml, 0.4 ml, 0.5 ml, 0.6 ml, 0.7 ml, 0.8 ml, 0.9 ml and 1 ml of each proteases to the same volume (10 ml) of milk (Guiama et al 2010).
Data was analyzed using SPSS version 16 software. One way ANOVA (Analysis of variance) used to test any significant difference (p<0.05) in clotting parameters (milk clotting time, curd yield and syneresis capacity) among different clotting proteases. Significant differences between means were set at 95 % (p<0.05) confidence interval.
The milk clotting activity of the plant extracts and calf rennet, expressed in clotting time (min), is shown in Table 1. Accordingly, the milk clotting time (MCT) of Ficus carica latex and Solanum incanum juice was nearly the same (about 11min) but was found to be significantly lower than that of Rhus natalensis and calf rennet (p<0.05) indicating a higher milk clotting activity. MCT obtained using crude juices of Solanum incanum (11.1±0.74 min) and Ficus carica latex (11.1±0.37 min) were less (higher MCA) than that of calf rennet (14.7±0.23 min). The MCA of Rhus natalensis extracts was found to be relatively inferior to the other plant extracts and to that of calf rennet indicating relatively low strength of particular coagulating enzyme (not specified at present study) found in the crude extract of the plant. Milk clotting time of Ficus carica latex (11.1±0.37) recorded in the present study was very close to the results of Bornaz et al (2010) who reported milk clotting time of 11 minutes.
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Table 1. Milk clotting time of Solanum incanum, Ficus carica, Rhus natalensis extracts compared with calf rennet |
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Type of coagulants |
N |
Milk clotting time (min*) |
Minimum |
|
Ficus carica latex |
4 |
11.1±0.37a |
10.6 |
|
Solanum incanum juice |
4 |
11.1±0.74a |
10.5 |
|
Rhus natalensis extracts |
4 |
36.5±1.18c |
35.1 |
|
Calf rennet |
4 |
14.7±0.23b |
14.5 |
|
p-value |
<.0001 |
||
|
Mean value in column with different superscripts are significantly different (p<0.05), SD = Standard deviation, *values represent (Mean) at constant temperature 32 ºC and pH 5.6 |
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The use of Ficus carica latex as a coagulant gave the highest curd yield (347.1 g/l of milk) compared to calf rennet (285.7±4.4) and the extracts from Solanum incanum juice and Rhus natalensis leaves (Table 2). The curd yields obtained from the action of Solanum incanum juice and Rhus natalensis extract were significantly inferior to that of rennet (p<0.05), making them less suited in terms of yield in cheese making compared to calf rennet. A similar finding was reported by Bornaz et al 2010 where the coagulum of fig latex was characterized by high yield (509.5 g/l of milk), a high loss level of fine particles, and a low syneresis capacity compared to that of Cynara extract and that of rennet. According to Low et al (2006) plant coagulants are characterized by excessive proteolytic activity leading to losses of peptides in the whey thus lowering cheese yields. Although Ficus carica latex gave the highest cheese yield; it had the lowest syneresis capacity (64g/l) compared to calf rennet and the extract of the other two plants (p<0.05). The higher yield obtained by Ficus carica latex could therefore be attributed to the high water retention by the coagulum. The wide variation in cheese yield obtained with the extracts of the three plants in the present study reflects the inherent difference in proteolytic activity between different species of plants.
Cheese yield is also influenced by factors such as milk composition, milk quality, heat treatment of milk, type of cheese and the processing methods used Formaggioni et al 2008). For instance, according to Abd El-Gawad and Ahmed (2011) , the fat content, the type of cheese and ripening time greatly influence the yield. Their results showed that a processed cheese and a Sudanese white cheese made out of 4.4% fat milk after 15 and 30 days ripening have shown higher yield of 2.85 kg and 1.75 kg and 2.75 kg and 1.5 kg respectively; While that made from 2.2 fat % yielded 2 kg and 1.25 kg and 2 kg and 1.2 kg, respectively using rennet as coagulant. In the present study, a milk fat % of 3.6 and protein % of 3.1 resulted to 347.1, 285.7, 206.1, and 250.7 g/l using Ficus carica latex, rennet, Rhus natalensis extract and Solanum incanum extract respectively.
Coagulum prepared from Rhus natalensis had the highest (88%) and that of Ficus carica latex had the lowest (64%) syneresis capacities (Table 2) indicating that the latter has the highest water holding capacity. The separation capacity of whey from curds produced by calf rennet and Solanum incanum juice was similar and fell between that of Rhus natalensis and Ficus carica.
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Table 2. Curd yield and syneresis capacity of bovine milk clotted by Solanum incanum juice, Ficus carica latex, Rhus natalensis extract and Calf Rennet. |
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Enzyme type |
Cheese yield(g/l) |
Syneresis capacity(g/l) |
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|
Mean ± SD* |
Minimum |
Maximum |
Mean ± SD* |
Minimum |
|
|
Solanum incanum extract |
251±5.4c |
246 |
256 |
82.3±1.1b |
81.1 |
|
Ficus carica latex |
347±7.1a |
339 |
353 |
64±5.8c |
58.1 |
|
Rhus natalensis extract |
206±9.5d |
200 |
217 |
88±1.8a |
86.2 |
|
Calf Rennet |
286±4.4b |
282 |
290 |
79.8±0.2b |
79.6 |
|
p-values |
<.0001 |
<.0001 |
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|
Mean values in column with different superscripts are significantly different (p<0.05), SD = Standard deviation, * Values represent (Mean) at constant temperature of 32 ºC and pH 5.6 |
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The mass of expelled whey with time revealed a high variability among the different coagulants (Figure 1). The pattern of change in mass of drained whey was similar in all the coagula tested (an initial increase in mass & then a plateau); but syneresis occurred at a relatively lower scale for Ficus carica latex indicating the coagulum obtained by the action of fig latex had the highest water retention capacity. This variability could probably be attributed to the differences in the physical properties of the gels produced by different plant coagulants (Bornaz et al 2010). According to Singh and Muthukumarappan (2008) , higher water retention or lower syneresis capacity of coagulum refers to the strength of gel network formed by different coagulants. The pattern of syneresis with time in the present study was generally similar with the findings of Bornaz et al (2010), who used fig latex, Cynara extract and calf rennet on ewe’s milk. However, while the syneresis capacity of calf rennet was almost the same (80%) with the present study, SC of fig latex in the present study (64%) was quite smaller than Bornaz et al (2010) findings (84%). Besides, unlike in the present study, the syneresis capacity of fig latex was higher than that of chymosin. The higher SC of the former study attributed to the difference on milk origin – cow’s vs ewe’s milk. McSweeney (2007) indicated that syneresis is directly related to casein content which is found in higher level in ovine than bovine milk.
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| Figure 1. Change of mass of drained whey and speed with time. |
The time required to coagulate 10 ml milk (Figure 2) has steadily decreased with increased concentration of coagulants with all three plants though the minimum and maximum time required has varied among the plant coagulants. In other words, as the amount of coagulants added to milk increased, the milk clotting activity increased in all three plants cases. The extremely high level clotting time range (15.1 to 36.5 min) exhibited by Rhus natalensis extracts indicated that the plant has lowest activity on milk. Using high concentration of Solanum incanum juice imparted a greenish color to the milk making its utilization inconvenient.
 |
| Figure 2.
The effect of volume of extract on time of clotting by Rhus
natalensis, Solanum incanum and Ficus carica latex (left) and Solanum incanum and Ficus carica latex (right) |
The authors acknowledge Mr. Tsehaye, a private commercial farmer in Adwa town, for his preliminary idea for this study and Mekelle University for financial and material support.
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Received 16 February 2018; Accepted 6 March 2018; Published 1 April 2018