Application of cultivable lactic acid bacteria isolated from Iranian traditional dairy products for the production of liquid and dried kashks

LWT - Food Science and Technology 116 (2019) 108519

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Application of cultivable lactic acid bacteria isolated from Iranian traditional dairy products for the production of liquid and dried kashks

T

Maryam Jafaria, Mohammad Rezaeia,b, Hamid Reza Gheisaric,1, Khadijeh Abharid, Gholamreza Jahed Khanikia,∗, Negin Noorib, Amin Mousavi Khaneghahe,∗∗ a

Department of Food Safety and Hygiene, School of Public Health, Tehran University of Medical Sciences, Tehran, Iran Department of Food Hygiene, Faculty of Veterinary Medicine, University of Tehran, Tehran, Iran c Department of Food Hygiene and Quality Control, School of Veterinary Medicine, Shiraz University, Shiraz, Iran d Food Sciences and Technology Department, National Nutrition and Food Technology Research Institute, Faculty of Nutrition Sciences and Food Technology, Shahid Beheshti University of Medical Sciences, Tehran, Iran e Department of Food Science, Faculty of Food Engineering, State University of Campinas (UNICAMP), Rua Monteiro Lobato, 80. Caixa Postal: 6121, CEP: 13083-862, Campinas, Sao Pulo, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Kashk Traditional dairy product Lactic acid bacteria Chemical composition Shelf life

This study was aimed to produce liquid and dried kashk as a traditional dairy-based product on an industrial scale. Furthermore, the microbial, chemical properties as well as the proximate composition including pH, moisture, salt, fat, protein, total volatile nitrogen (TVN) and peroxide value (PV) were assessed after 1, 10 and 20 days of cold storage (7 °C). Also, the samples from traditional dairy products such as yogurt, doogh, dried kashk, and lolek were collected, and the diversity of lactic acid bacteria (LAB) was determined as L. lactis subsp cremoris, Streptococcus thermophilus, L. lactis subsp lactis, Lactobacillus plantarum, Lactobacillus helveticus, Lactobacillus bulgaricus, Lactobacillus casei, and Lactobacillus fermentum. The mean values of fat, protein, and TVN for dried and liquid kashk samples were measured as (7.4 ± 0.7, 2.7 ± 0.1%), (51 ± 3.8, 8.7 ± 0.6 g/100 g) and (15.3 ± 3.9, 14.9 ± 4.1 mg/100 g), respectively. The microbial analysis revealed yeasts and molds were the most prevalent microorganisms in both types of kashks, while no Coliform, Escherichia coli, and Staphylococcus aureus bacteria were found in all samples. In conclusion, the selected species from traditional products which presented a specific property may provide a wide range of genetic variants to design modified species with desired characteristics.

1. Introduction The diversity in dairy product's market is increasing due to their beneficial impacts on human health as well as newly introduced formulations (Hashemi et al., 2017; Mahmood Fashandi, Abbasi, & Mousavi Khaneghah, 2018; Mousavi Khaneghah, D Chaves, & Akbarirad, 2017). The most important factors that influence the acceptability of these products are their positive health effects, desirable nutritional trait, favored sensory characteristics and acceptable shelflife (Rahimirad, 2014; Soltani & Güzeler, 2013). The producing of fermented dairy products due to the high levels of nutritional values as well as efficiency in the prevention of some diseases are points of interest for both academic and industrial sectors (Campagnollo et al., 2016). However, their features could be varied in

different regions mainly depends on the used microbial cultures (Karimpour, Boyerahmad, & Yasuj, 2013). In this regard, several types of fermented dairy products were introduced around the world (Savadogo et al., 2004).In Iran, various products such as doogh, yogurt, cashew, gharaghooroot, and cheese are associated with cultural diversity (Ebrahimi, Ouwehand, Hejazi, & Jafari, 2011). Among them, Kashk as a fermented dairy product is produced either in dry form (traditionally) or liquid form (industrially) (Meybodi, Ebrahimi, & Mortazavian, 2016). The dried kashk, which is originated from the Middle East is a round shape and walnut-size product (Sadrizadeh, Khezri, Dehghan, & Mahmoudi, 2018) and can be stored for a long time at room temperature without deterioration or lose in the nutritional values (Dehkordi, Yazdani, Mozafari, & Valizadeh, 2014; Soltani and Güzeler, 2013; Tavakoli et al., 2009). The liquid form of the

∗

Corresponding author. Corresponding author. E-mail addresses: [email protected] (G.J. Khaniki), [email protected] (A. Mousavi Khaneghah). 1 Died 28 DEC 2017. Deceased. ∗∗

https://doi.org/10.1016/j.lwt.2019.108519 Received 3 May 2019; Received in revised form 5 August 2019; Accepted 20 August 2019 Available online 20 August 2019 0023-6438/ © 2019 Elsevier Ltd. All rights reserved.

LWT - Food Science and Technology 116 (2019) 108519

M. Jafari, et al.

2.3. Genotypic identification

kashk is manufactured from yogurt or dried kashk in industrial units (Soltani & Güzeler, 2013). Both forms; liquid and dried contain ahigh amount of proteins and some minerals such as calcium, potassium, vitamins group B and organic acids (lactic acid) (Soltani & Güzeler, 2013). The flavor, aroma, and texture of the kashk are influenced by several factors, particularly enzymatic activities of LAB as main microflora used among dairy products (Barnali & Subhankar, 2010; Sun et al., 2010). Hence, it is necessary to recover novel starter cultures mainly LAB; from both traditional dairy products and non-dairy sources (Azadnia & Khan Nazer, 2009; RoushanZadeh, Eskandari, Shekarforoush, & Hosseini, 2014). In this context, the isolation of LAB from traditional dairy products for using in yogurt and cheese production has been investigated in previous studies (Fguiri, Ziadi, Rekaya, Samira, & Khorchani, 2017; González, Cuadrillero, Castro, Bernardo, & Tornadijo, 2015). However, based on our knowledge, no investigation was conducted to retrieve the LAB from kashk as one of the favored dairy-based products in the Middle East region. Therefore, the current study was aimed to identify phenotypic and genotypic diversity of dominant LAB isolated from traditional dairy products of several tribes in Fars province and their incorporation into liquid and dried kashks and further evaluate microbial, chemical and organoleptic characteristics of produced kashk samples.

The genomic of DNA was extracted from bacterial isolates by phenol extraction method. For DNA amplification 5 μL genomic of isolated DNA, 15 μL PCR buffer, 3 μL dNTPs (10 mM) (Fermentas, Lithuania), 9 μL MgCl2 (50 mM), 0.5 μL Taq polymerase 5 IU/μL, 3 μL of 1200-bp primer 16S-FA 5′-AGAGTTTGATCCTGGCTCAG-3′and 16SRA5 ′-AGGAGGTGZTCCAGCCGC-3'. DNAase free H2O (CinnaGen Inc., Iran) were mixed and reached to a final volume of 50 μL. DNA amplification was carried out with a thermal cycler programmed as follows: an initial denaturation of 3 min at 94 °C, 35 cycles (consisting of denaturation 45 s at 94 °C, annealing 30 s at 58 °C, 30 s at 52 °C, and polymerization 5 s at 72 °C), followed by a final extension at 72 °C for 5 min. The PCR products were electrophoresed on 1.0% agarose gels, stained with Ethidium bromide and photographed (RoushanZadeh et al., 2014). 2.4. 16S rDNA sequencing The 1200-bp PCR products were purified from agarose gels using a HiYield™ gel extraction kit (Bioneer, South Korea). Then, 500 μL digestion solutions (DF buffer) were mixed with the PCR products and incubated at 50 °C until complete digestion of gel. The aqueous phase was transferred into a plasmid purification column and centrifuged 1500 g for 9 min. Then, it was transferred into an upper column and centrifuged 1500 g for another 9 min. Afterward, was mixed with 400 μL of washing buffer and centrifuged at 14,000 g for 2 min. Finally, the aqueous phase was removed, and 600 μL of plasmid washing solution (80% ethanol) was added into the upper column and the tubes centrifuged at 14,000 g for additional 2 min, and the aqueous phase was removed once more. The upper column was transferred into a new tube where 75 μL DNAase free water was added and centrifuged at 14,000 g for 2 min. The recovered PCR amplicons were placed for sequencing analysis (CinnaGen Inc., Iran) (Liu et al., 2012; RoushanZadeh et al., 2014; Wang et al., 2016). DNA sequence analysis was assembled and edited using the BioEdit sequence alignment editor version 5.0.9. All sequences were deposited in GenBank.

2. Materials and methods 2.1. Sample collection Sixty samples (no = 15 samples from each product) of traditional milk products including traditional yogurt, doogh (Iranian yogurt-based drink), dried kashk and lolek (semi-dried kashk) were collected from the villages of the Fars province, Iran (Summer 2017). Fifteen samples also were taken from industrial liquid kashk of Fars dairy units from April to December 2017. The samples were kept at 4 °C and analyzed up to 24 h.

2.5. Application of isolated LAB into the kashk Four strains of LAB were selected and used as a starter culture for fermenting the milk to produce kashkincluding L.casei, L.plantarum, L.helveticus, and S. thermophilus. These strains were selected based on their predominance in traditional spontaneous kashk fermentation and also their desirable properties such as rapid-acidification, high proteolytic and low lipolytic activities, ability to produce exopolysaccharides (EPS), and has antimicrobial properties.

2.2. Isolation and biochemical identification The samples were cultured on Plate Count Agar medium (Merck, Germany) at 30 °C for aerobic mesophilic bacteria. De Man Rogosa Sharpe (MRS) agar (pH = 5.7) supplemented with 1% skim milk (Merck, Germany) was used for isolation of lactobacilli. Additionally, M17 agar (pH 7.15) supplemented with 1% lactose (Himedia, Hindi) was used in order to isolate streptococci, lactococci, and enterococci. Also, the culture media was supplemented with 50 mgL−1 of Natamycin to prevent the mold and yeasts contamination (Botes, Todorov, Von Mollendorff, Botha, & Dicks, 2007). MRS and M17 plates were incubated under aerobically and anaerobically conditions, respectively, using the gas pack system (Merck Anaerocult Type A) at 30, 37 ̊ and 42 ̊°C (Abd El Gawad, Abd El Fatah, & Al Rubayyi, 2010; Maqsood, Hasan, & Masud, 2013; Terzic-Vidojevic et al., 2009). The colonies were subjected to gram reaction, catalase production, oxidase activity, spore formation, and cell morphology (color, shape, and size). Isolates of gram-positive, catalase and oxidase negative, non-spore, cocci or rod were selected as a presumptive LAB. For further identification following tests were performed: growth at 10, 40 and 45 °C; growth ability at 2, 4 and 6.5% NaCl concentrations; growth at pH 9.6; Arginine hydrolysis and gas production from citrate; reduced Methylene Blue; gas production from glucose; ability to ferment D Mannitol, D (−) Raffinose, D (−) Ribose, D (+) Galactose, Maltose, Lactose, L (+) Arabinose, glucose, D (+) Xylose, D (−) Salicin, D (+) Mannose, sucrose, Fructose and Rhamnose (Abd El Gawad et al., 2010; González et al., 2015; Reginensi, Gonzalez, & Bermudez, 2013).

2.6. Biomass production First, isolates were sub-cultured on MRS and M17 broth at 37 °C for 16 h. One hundred mL of the medium were inoculated, with 10% of the active culture. The optical density at 600 nm (OD 600) was measured in order to monitor the bacterial growth. The growing amount was measured based on the difference between the initial OD and the final OD at which cells were collected (ΔOD). The slope of the linear part of the curve representing Log OD versus time was taken as the indicator of the maximum growth rate. At the initial stationary phase, 30 mL of culture was harvested by centrifugation at 7500–8000 g for 15 min at 4 °C and the supernatant was removed. Following steps are completely performed in the sterile condition for purification. Ten mL of sterile normal saline or peptone water was added into the bacterial cell, and the tubes were centrifuged at 7500–8000 g for 10 min after removing of the aqueous phase, the previous steps were repeated three times. The biomass pellets were suspended in sterile reconstituted skim milk and stored at 4 °C, and finally were used in less than 16 h (Ayad, Nashat, ElSadek, Metwaly, & El-Soda, 2004). Monitoring of OD was performed 2

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aureus was counted by surface plating on Baird Parker Agar (BPA) and incubating at 37 °C for 24–48 h (Azadnia & Khan Nazer, 2009).

Table 1 The bacterial species used in the samples. Kashk samples

Strains

I II III IV V VI VII control

S. S. S. S. S. S. S. S.

2.10. Statistical analysis thermophilus thermophilus thermophilus thermophilus thermophilus thermophilus thermophilus thermophilus

+ + + + + + + +

L. L. L. L. L. L. L. L.

plantarum casei plantarum + L. casei helveticus + L. casei + L. plantarum plantarum + L. helveticus helveticus + L. casei helveticus delbrueckii ssp. bulgaricus

All experiments were carried out in triplicate. All analyses were performed using SPSS software version 21. Data were subjected to the ttest, one-way analysis of variance (ANOVA) and Duncan's multiple range tests to determine significant differences between means. Statistical significance was set at P < 0.05. 3. Results and discussion

during bacterial growth in a broth culture medium, which allowed estimation of the maximum growth rate. The fermentation broth was centrifuged, and the pellet was used to determine biomass.

3.1. Isolation of LAB A total of 330 dominant isolates were collected from 75 samples and purified using PCA, MRS and M17 agar media.

2.7. Production of kashk samples

3.2. LAB phenotypic and biochemical identification

The milk with 4% fat was employed for yogurt production and checked for presences of antibiotics, then were filtered and homogenized. The milk was pasteurized at 90–95 °C for 5–10 min based on the heat treatments of the dairy industry. The pasteurized milk was cooled to 43 °C and inoculated with the starter culture. A total of seven different kashk samples was produced using a mix of confirmed isolates (Table 1). Approximately 10 mL of starter cultures were inoculated to each batch. This was determined by the initial viable counts of yogurt which was about 107 CFU/mL in each batch and furthermore was measured by spread plate count method and incubated at 42 °C until the pH reached 4.6 (about 4–4.5 h). The ratio of the bacilli to cocci in yogurt starter culture was adjusted as 1:1. The starter cells were inoculated to milk by stirring and incubated at 42–43 °C for 3–4 h. Once the pH decreases to 4.5, yogurts were cooled to 5 or 10 °C and stored for 3 days. The yogurts were homogenized (50–55 °C, 2 bar) and whey powder (1–2%), water and edible salt were added and heated at 80–90 °C for 2.5–3 h. To produce liquid kashk, samples were packaged, and dry kashks were rounded after cooling and leaving for dry among several days depending on the thickness of the kashk. The final products were packaged and stored at 7 °C. The storage time is based on the expiry date of the industrial samples.

From 330 g-positive isolates obtained from dairy products, 157 (47.57%) were negative for catalase and oxidase activity, non-spore forming bacteria (Enterococcus sp. (2.25%), Lactococcus sp. (20.3%), Lactobacillus sp. (53.50%) and Streptococcus sp. (40.6%)) and all belonging to the LAB family (Table 2). The predominant LAB isolates and the distribution of their species in various traditional dairy products was shown Table 3 and Table 4, respectively. However, predominant LAB were different among dairy samples. In this research, the number of isolates from yogurt and doogh was higher than lolek samples. Especially from dried kashk and industrial liquid kashk a few genera of LAB have been isolated and identified. Diversity of bacterial species can be associated with the composition of raw milk and animal breeds, indigenous regional effects, type of culture medium used, the salt concentration in the environment as well as the presence of calcium. 3.3. Genotypic identification In order to have more accurate identification regarding the LAB species, PCR assays (Fig. 1) were used. Eleven isolates (L. lactis spp cremoris, S. thermophiles, L. lactis spp lactis, L. plantarum, L. heleviticus, L. Lactobacillus delbruecki subsp. bulgaricus, L. plantarum, L. casei, L. fermentum) (Table 5) were identified by 16S rDNA sequences and deposited in the NCBI nucleotide sequence databases.

2.8. Chemical analysis The chemical analysis of the kashk samples was performed on day 1, 10, and 20. The pH values of kashk samples were measured using a pH meter (Knick- Germany) according to AOAC guidelines (AOAC, 1990). To determine the moisture content, the oven drying method was used (Electronic Fater Feb 50 L), which was adjusted as 102 ± 2 °C. Fat percent and total protein content of kashk samples were measured by Gerber and Kjeldahl's methods, respectively (Bonczar, Wszołek, & Siuta, 2002). The salt concentration was determined according to the method described in AOAC guidelines (AOAC, 1990). Determination of PV and TVN was done by iodometric titration and Kjeldahl's methods, respectively, according to AOAC guidelines (AOAC, 1990).

3.4. Application of isolated LAB in the preparation of kashk In this study, four functional isolates were used which showed high acidification, high proteolytic capacity and high EPS production. One of the most significant criteria for selecting a starter culture was the acidification velocity (Ma et al., 2012). The thermophilic strains, S. thermophilus and L. helveticus produced LA after 6 h of incubation. The level of acidity due to these strains increased during incubation especially for S. thermophiles. In fact, these strains presented very high and fast acidification capacity (Badis et al., 2004; Omafuvbe & Enyioha, 2011). Marroki, Zúñiga, Kihal, and Pérez-Martínez (2011) reported that Table 2 Number of LAB isolated from various dairy samples using PCA, MRS and M17 culture media.

2.9. Microbial analysis Ten grams of each kashk sample were weighed aseptically and homogenized with 90 mL of sterile Ringer's solution. To enumerate Coliforms and Escherichia coli in the samples, Violet Red Bile Agar (VRBA) and Lauriyel Sulfate Tryptose were used and incubated at 37 °C for 24 h. Also, the Yeast Extract Dextrose Chloramphenicol Agar (25 °C for 5 days) was used to enumerate molds and yeasts. Staphylococcus 3

LAB

Yogurt

Doogh

Lolek

Dried kashk

Liquid kashk

Total

Cocci Rod Total

29 50 79

21 17 38

14 6 20

20 – 20

– – –

84 73 157

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Table 3 Distribution of LAB species in various traditional dairy products in Fars Province. Species

Yogurt

Doogh

Lolek

Dried kashk

Liquid kashk

S. thermophilus S. intestinalis E. fecalis L. lactis spp lactis L. lactis spp cremoris L. lactis L. plantarum L. brevis L. helveticus L. delbrueckii L. bulgaricus L. casei L. fermentum L.acidophilus

+ – + + + + + + + + + + + +

+ – – + + + + + + + + + + +

+ – + + + – + – + – + – – –

+ + – – – – – – – – – – – –

-

Table 5 Species identification by sequencing of the 16s rDNA.

The percentage of lactic acid bacteria isolated from collected samples

S. thermophilus S. intestinalis E. fecalis L. lactis spp lactis L. lactis spp cremoris L. lactis L. plantarum L. brevis L. helveticus L. delbrueckii L. bulgaricus L. casei L. fermentum L.acidophilus L. casei subsp pseudoplantarum

29.93% 4.45% 1.9% 9.55% 7.64% 1.9% 11.46% 1.9% 5.09% 6.36% 8.91% 4.45% 1.9% 2.54% 1.9%

Species

Similarities (%)

Ls13 Kb27 Yf52 Yb16 Dsr45 Db22 Yb24 Dsh14 Yf45 Yb27 Yf44

Lactococcus lactis spp cremoris Streptococcus thermophilus Lactococcus lactis spp lactis Streptococcus thermophilus Lactococcus lactis spp lactis Lactobacillus plantarum Lactobacillus helveticus Lactobacillus bulgaricus Lactobacillus plantarum Lactobacillus casei Lactobacillus fermentum

99% 100% 99% 100% 100% 97% 98% 95% 97% 98% 95%

bulgaricus, L. casei, L. jugurti and L. plantarum while tripeptide was presented only in L. casei and L. plantarum (Ayad et al., 2004). 3.5. Biomass production and growth rate

Table 4 Phenotypic identification of LAB strains isolated from Iranian dairy products. Isolated strains according to phenotypic methods

Isolate

The results revealed that L. plantarum and L. casei species were grown faster and reached the stationary phase in 8 h, while S. thermophilus and L. helveticus produced low amounts of biomass and reached the stationary phase slowly. The value of ΔOD 600 was found in a range of 0.3–1 among all tested bacteria, while theL. plantarum had the excellent mean value of 0.8–1. In a study by Ayad et al. (2004), based on the biomass, cultures was divided into three groups in the range of < 0.6 - ≥ 1.33 and L. rhamnosus, L. delbrucki subsp. Bulgaricus, L. plantarum, and L. lactis subsp. cremoris exhibited the highest biomass production while compared with other species. The low yields of biomass could be a result of the poor separation of biomass during centrifugation, due to the production of exopolysaccharides (EPS) that inhibit separation of bacterial cells from the culture medium. 3.6. Chemical analysis Comparison of analytical results obtained from the pH value of dried and liquid kashk samples were presented in Fig. 2 and Fig. 3. As it was expected, a decrease in pH of all samples was observed during storage time, while no significant (P > 0.05) difference in the pH value between the samples during the time was noted. The mean pH of kashk samples were affected by pH of basic raw material (yogurt), heat treatment and principally the starter bacteria, as S. thermophiles which showed a high and fast acidifying capacity, whereas L. Plantarum and L. casei species presented low acidification capacity (Ma et al., 2012; Omafuvbe & Enyioha, 2011). The fast acidifying LAB is a good candidate for primary starters in the dairy fermentation, while the poor acidifier species depending on their other important properties, and could be applied as adjunct cultures (Ayad et al., 2004).

L. plantarum subgroups presented distinct behaviors regarding the acidification (Marroki et al., 2011). According to Medina, Oliszewski, Mukdsi, Van Nieuwenhove, and González (2011), L.acidophilus, L. paracasei, L. plantarum, L. sakei and helveticus strains may have moderate lipase and esterase activities (Medina et al., 2011). However, Lactobacillus strains showed adequate technological properties as a starter culture (Landeta, Curiel, Carrascosa, Muñoz, & De Las Rivas, 2013). The proteolytic system during casein utilization provides essential amino acids for cells during growth in milk which also has industrial importance due to its contribution to the development of the organoleptic properties of fermented milk products (Savijoki, Ingmer, & Varmanen, 2006). The presence of amino peptidases and dipeptides also were correlated to activity of L. helveticus, L. acidophilus, L. lactis, L.

Fig. 1. Examples of RAPD-PCR profiies obtained from LAB isolated with oligonucleotide primer. M: Marker 1200: The length of amplification products. 4

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Fig.2. Physical properties of dried kashk samples during 20 days storage at 7 °C (Mean). Values not sharing the same superscript are significantly different (P < 0.05). Bars represent standard error values.

the PV test, were displayed in Tables 6 and 7 for dried and liquid kashk, respectively. In this regard, a significant difference (P < 0.05) in terms of PV among liquid and dried kashks during the storage period was observed. For both kashk types, during the first storage days, a slight increase in TVB-N level was noted, which could reflect the amines production during autolytic processes as well as contamination by mold and yeast.

Some physical properties of dried and liquid samples, respectively, were demonstrated in Figs. 2 and 3. One of the key element which contributes to taste, safety, and consistency and yield properties of foods is salt while no significant difference (P > 0.05) in the concentration of salt between the samples was noted. The changes in the chemical composition of dried and liquid kashk samples were presented in Tables 6 and 7, respectively. Although the initial fat content of raw material was quite similar, a significant difference in the fat content (P < 0.05) between dried and liquid kashks samples was noted which can be associated with the lipolytic activity of used species as starter cultures (Medina et al., 2011). Additionally, a significant difference (P < 0.05) regarding protein content between liquid and dried kashks during the storage period (Except for sample IV in dried kashk) was observed which might be attributed to lipolytic and proteolytic activities of molds and yeasts (Lavoie, Touchette, St-Gelais, & Labrie, 2012). The quality of kashk samples, which was evaluated by

3.7. Microbial analysis The obtained results regarding the microbial analysis for kashk samples showed that no Coliform, Escherichia coli and Staphylococcus aureus bacteria were found in all samples during the storage period while molds, and yeasts were the most prevalent microorganisms as less than 10 CFU/g. According to the national standard of Iran, the number of molds and yeast in industrial kashk should be less than 100 CFU/g 5

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Fig. 3. Physical properties of liquid kashk samples during 20 days storage at 7 °C (Mean). Values not sharing the same superscript are significantly different (P < 0.05). Bars represent standard error values.

Thermal process during the production and low water activity of kashk are also among the factors that strictly inhibit microorganism's growth. In other hands, the molds and yeasts are widely distributed in nature, and under suitable conditions of moisture, salts, and temperature they can grow on almost any type of food during processing, packaging or storage from raw to ready product, to become a serious issue for most food products. Drying open-air (for dried kashk), different fermentation time, staple, the traditional way of production which might contaminate the product with the flora of the skin of the producer are important sources of microbiological contamination (Mashak et al., 2014). Lack of hygienic condition during the handling of the product to the market, especially in traditional products, can provide favored conditions for post-contamination (Dehkordi et al., 2014; Habibi & Adabi, 2015).

(ISIRI, 2007, p. 10154). There are many factors that restrict the growth of pathogenic bacteria in the matrix of kashk. Salt is a principle additive in producing kashk that besides enhancing the flavor, can strongly inhibit bacterial survival through plasmolysis phenomenon. Although there are some reports regarding detection of halophile bacteria such as S. aureus in kashk. Lactobacilli which are used as starter have the ability to compete with other bacteria through production of lactic acid (LA) and bacteriocin; therefore, incorporation of LAB in this product improves the microbial safety. However, the degree of inhibition of pathogenic bacteria by LAB is highly strain-depended; many of them play an important role in preservation of fermented foods. Decreasing in pH as the result of LAB proliferation and metabolism lead to a harsh environment for both pathogenic and spoilage bacteria. Even the growth of LAB restricted while pH drop and therefore, yeasts and mold become the dominant flora of fermented foods (Mashak, 2016; Mashak, Sodagari, Mashak, & Niknafs, 2014). 6

56.2 ± 0.28BCc 52.65 ± 0.21Ac 55.05 ± 0.63ABCc 54.4 ± 3.39ABb 57.95 ± 1.06Cc 53.55 ± 1.06ABb 58.4 ± 0.84Cc 68.31 ± 0.96Dc

7.93 ± 0.04Ac 8.48 ± 0.11BCc 7.88 ± 0.1Ac 7.9 ± 0.03Ac 8.3 ± 0.14Bc 8.6 ± 0.01Cc 8.59 ± 0.007Cc 9.6 ± 0.14Dc

1 2 3 4 5 6 7 C

9.2 ± 0.14Ba 7.6 ± 0.14Aa 10.4 ± 0.14Ca 11.8 ± 0.14Da 11.6 ± 0.14Da 14.4 ± 0.14Ea 11.6 ± 0.14Da 14.94 ± 0.07Fa

TVN mg/100 g

3.1 ± 0.42Ba 2.55 ± 0.21ABa 2.1 ± 0.42Aa 2.6 ± 0.28ABa 2.9 ± 0.14ABa 3.05 ± 0.35Ba 3.15 ± 0.49Ba 4.1 ± 0.14Ca

PV mEq/Kg

7.24 ± 0.04Ab 7.75 ± 0.07Cb 7.18 ± 0.1Ab 7.23 ± 0.07Ab 7.63 ± 0.04Cb 7.42 ± 0.02Bb 7.6 ± 0.07Cb 8.83 ± 0.04Db

Fat %

Day 10

51.25 ± 0.35CDb 48.25 ± 0.35Ab 50.3 ± 0.42BCb 50.45 ± 0.77BCab 52.4 ± 0.56Db 48.75 ± 1.06ABa 52.45 ± 0.63Db 63.1 ± 1.26Eb

Protein g/100 g

14.15 ± 0.21Bb 11.2 ± 0.98Ab 16.2 ± 0.14CDb 17.25 ± 0.07EFb 16.6 ± 0.28DEb 17.45 ± 0.35EFb 15.3 ± 0.14Cb 18.05 ± 0.07Fb

TVN mg/100 g

3.4 ± 0.14ABa 3.3 ± 0.42ABa 2.8 ± 0.14Aa 3.25 ± 0.35ABa 3.85 ± 0.21Bb 4 ± 0.42Ba 3.65 ± 0.49Ba 5.4 ± 0.14Cb

PV mEq/Kg

6.55 ± 0.06Aa 6.99 ± 0.01Ba 6.6 ± 0.07Aa 6.6 ± 0.13Aa 6.76 ± 0.02Aa 6.58 ± 0.04Aa 6.59 ± 0.007Aa 8.15 ± 0.21Da

Fat %

Day 20

47.25 ± 0.35BCa 44.9 ± 0.14Aa 46.4 ± 0.56ABCa 47 ± 1.41BCa 49.55 ± 0.35Da 46.25 ± 0.35ABa 48.2 ± 0.28CDa 54.01 ± 1.4Ea

Protein g/100 g

18.95 ± 0.07Bc 16.6 ± 0.28Ac 19.8 ± 0.28Cc 20.95 ± 0.07Dc 20.75 ± 0.35Dc 20.9 ± 0.14Dc 19.4 ± 0.14BCc 21.35 ± 0.49Dc

TVN mg/100 g

5.2 ± 0.28Ab 5.03 ± 0.03Ab 5.15 ± 0.21Ab 5.25 ± 0.35Ab 5.44 ± 0.19Ac 5.31 ± 0.43Ab 5.26 ± 0.36Ab 6.6 ± 0.28Cc

PV mEq/Kg

7

9.65 ± 0.07Ea 8.64 ± 0.04CDa 9.55 ± 0.07Ea 8.45 ± 0.07Ba 8.53 ± 0.1BCa 8.0 ± 0.07Aa 8.7 ± 0.05Da 8.1 ± 0.13Aa

2.85 ± 0.07Ca 2.82 ± 0.02Cba 2.85 ± 0.07Ca 2.7 ± 0.007Ba 2.62 ± 0.04ABa 2.58 ± 0.04Aa 2.59 ± 0.07ABa 2.45 ± 0.07Aa

1 2 3 4 5 6 7 C

20.28 ± 0.07Da 16.35 ± 0.21Ca 12.15 ± 0.21BAa 11.25 ± 0.07Ba 11.01 ± 0.01Aa 20.1 ± 0.9Da 11.05 ± 0.07Aa 11.6 ± 0.14Bb

TVN mg/100 g

1.9 ± 0.42ABa 1.35 ± 0.21ABa 1 ± 0.28Aa 1.4 ± 0.28ABa 1.8 ± 0.28ABa 1.9 ± 0.42ABa 2 ± 0.56Ba 1.2 ± 0.14Aa

PV mEq/Kg

2.85 2.82 2.85 2± 2.62 2.58 2.59 2.45

Fat % ± 0.07Ca ± 0.02Cba ± 0.07Ca 0.007Ba ± 0.04ABa ± 0.04Aa ± 0.07ABa ± 0.07Aa

Day 10

9.7 ± 0.06Ca 8.61 ± 0.07Ba 9.52 ± 0.04Ca 8.44 ± 0.04Ba 8.13 ± 0.3Aa 8.1 ± 0.13Aa 8.47 ± 0.24Bba 7.93 ± 0.07BCa

Protein g/100 g

20.85 ± 0.07Da 16.74 ± 0.08Ab 12.55 ± 0.07Ba 11.55 ± 0.07Aab 11.3 ± 0.14Ab 20.65 ± 0.91Cb 11.31 ± 0.02Aab 12.55 ± 0.07Ba

TVN mg/100 g

1.9 ± 0.42ABa 1.3 ± 0.28ABa 1.1 ± 0.14Aa 1.5 ± 0.28ABa 1.85 ± 0.21Ba 2 ± 0.28Ba 2.1 ± 0.56Aa 1.35 ± 0.21ABa

PV mEq/Kg

2.76 2.84 2.75 2.62 2.57 2.54 2.45 2.32

Fat % ± ± ± ± ± ± ± ±

Day 20

0.07Da 0.06DCa 0.07Da 0.02CBa 0.04Ba 0.05ABa 0.07Aa 0.03Aa

9.34 ± 0.05Ea 8.6 ± 0.14Da 9.45 ± 0.07Ea 7.93 ± 0.07BCa 8.28 ± 0.03ABa 7.51 ± 0.01Aa 8.25 ± 0.3CDa 7.5 ± 0.72Aa

Protein g/100 g

20.95 ± 0.07Da 16.75 ± 0.07Cc 12.55 ± 0.07Ba 11.6 ± 0.14Bb 11.35 ± 0.21Ac 20.7 ± 0.98Dc 11.4 ± 0.14Ab 16.35 ± 0.21Ca

TVN mg/100 g

2 ± 0.42ABa 1.4 ± 0.2Aa 1.2 ± 0.14Aa 1.8 ± 0.2ABa 2.1 ± 0.2ABa 2.25 ± 0.35Ba 2.28 ± 0.4Ba 1.5 ± 0.28ABa

PV mEq/Kg

Different lowercase letters in the same row indicate significant differences (P < 0.05) among each sample, whereas different capital letters in the same column indicate significant differences (P < 0.05) between samples analyzed. TVN: Total Volatile Nitrogen. PV: Peroxide Value. C:Control.

Protein g/100 g

Fat %

No

Day 1

Table 7 Chemical composition changes of liquid kashk samples during 20 days storage at 7 °C (Mean ± SD).

Different lowercase letters in the same row indicate significant differences (P < 0.05) among each sample, whereas different capital letters in the same column indicate significant differences (P < 0.05) between samples analyzed. TVN: Total Volatile Nitrogen PV: Peroxide Value. C:Control.

Protein g/100 g

Fat %

No

Day 1

Table 6 Chemical composition changes of dried kashk samples during 20 days storage at 7 °C (Mean ± SD).

M. Jafari, et al.

LWT - Food Science and Technology 116 (2019) 108519

LWT - Food Science and Technology 116 (2019) 108519

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4. Conclusion

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Due to the rapid industrialization, kashk as a popular Iranian dairy product needs to be produced with better quality and modified flavor. Therefore, in the current investigation, kashk were produced using LAB species isolated from traditional dairy products while these bacteria play a principal role in increasing the quality of samples. In this study, after 20 days of cold storage samples had good quality, however, chemical and microbial quality of kashks decreased. Therefore, it was concluded that isolated LAB can be used as starter cultures or adjuncts in industrial dairy products and the optimal exploitation of them under specific. In conclusion, the selected specific LAB species may provide products with traditional-industrial scale properties, while their proper use requires special conditions and further studies. The selected species from traditional products which presented a specific property may provide a wide range of genetic variants for the design of genetically modified species with desired characteristics. Conflicts of interest The authors declare that there is no conflict of interest. Acknowledgements This article was donated in memoriam of Dr. Hamid Reza Gheisari, who passed away during the experimental phase of this manuscript. This study was part of an MSc Thesis supported by the School of Public Health, Tehran University of Medical Sciences and Health Services (grant no. 240/426). References Abd El Gawad, I., Abd El Fatah, A., & Al Rubayyi, K. (2010). Identification and characterization of dominant lactic acid bacteria isolated from traditional rayeb milk in Egypt. Journal of American Science, 6, 728–735. AOAC (1990). Official methods of analysis (3rd ed.). Washington, D.C: Association of Official Analytical Chemists. Ayad, E., Nashat, S., El-Sadek, N., Metwaly, H., & El-Soda, M. (2004). Selection of wild lactic acid bacteria isolated from traditional Egyptian dairy products according to production and technological criteria. Food Microbiology, 21, 715–725. Azadnia, P., & Khan Nazer, A. (2009). Identification of lactic acid bacteria isolated from traditional drinking yoghurt in tribes of Fars province. Iranian Journal of Veterinary Research, 10, 235–240. Badis, A., Guetarni, D., Moussa-Boudjemaa, B., Henni, D., Tornadijo, M., & Kihal, M. (2004). Identification of cultivable lactic acid bacteria isolated from Algerian raw goat's milk and evaluation of their technological properties. Food Microbiology, 21, 343–349. Barnali, A., & Subhankar, P. (2010). Isolation and characterization of lactic acid bacteria from dairy effluents. Journal of Environmental Research and Development, 4, 983–991. Bonczar, G., Wszołek, M., & Siuta, A. (2002). The effects of certain factors on the properties of yoghurt made from Ewe's milk. Food Chemistry, 79, 85–91. Botes, A., Todorov, S. D., Von Mollendorff, J. W., Botha, A., & Dicks, L. M. (2007). Identification of lactic acid bacteria and yeast from boza. Process Biochemistry, 42, 267–270. Campagnollo, F. B., Ganev, K. C., Khaneghah, A. M., Portela, J. B., Cruz, A. G., Granato, D., et al. (2016). The occurrence and effect of unit operations for dairy products processing on the fate of aflatoxin M1: A review. Food Control, 68, 310–329. Dehkordi, F. S., Yazdani, F., Mozafari, J., & Valizadeh, Y. (2014). Virulence factors, serogroups and antimicrobial resistance properties of Escherichia coli strains in fermented dairy products. BMC Research Notes, 7, 217. Ebrahimi, M., Ouwehand, A., Hejazi, M., & Jafari, P. (2011). Traditional Iranian dairy products: A source of potential probiotic lactobacilli. African Journal of Microbiology Research, 5, 20–27. Fguiri, I., Ziadi, M., Rekaya, K., Samira, A., & Khorchani, T. (2017). Isolation and characterization of lactic acid bacteria strains from raw camel milk for potential use in the production of yogurt. Journal of Food Science and Nutrition, 3, 1–8. González, L., Cuadrillero, A. F., Castro, J. M., Bernardo, A., & Tornadijo, M. E. (2015). Selection of lactic acid bacteria isolated from San Simón da Costa Cheese (PDO) in order to develop an autochthonous starter culture. Advances in Microbiology, 5, 748. Habibi, N., & Adabi, T. (2015). Determination of some chemical charcteristics dry Kashk in Kurdistan province of Iran. International Journal of Scientific Engineering and Applied Science, 1, 441–443. Hashemi, S. M. B., Mousavi Khaneghah, A., Kontominas, M. G., Eş, I., Sant'Ana, A. S.,

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