Psychrotrophic Bacteria in Foods Disease and Spoilage - Food Trade Review
J Zhejiang Univ Sci B. 2018 Aug; nineteen(8): 630–642.
Spoilage potential of psychrotrophic bacteria isolated from raw milk and the thermo-stability of their enzymes*
Received 2017 Jul 10; Accepted 2017 Dec 28.
Abstract
The storage and transportation of raw milk at low temperatures promote the growth of psychrotrophic leaner and the production of thermo-stable enzymes, which pose dandy threats to the quality and shelf-life of dairy products. Though many studies have been carried out on the spoilage potential of psychrotrophic leaner and the thermo-stabilities of the enzymes they produce, further detailed studies are needed to devise an constructive strategy to avoid dairy spoilage. The purpose of this study was to explore the spoilage potential of psychrotrophic leaner from Chinese raw milk samples at both room temperature (28 °C) and refrigerated temperature (7 °C). Species of Yersinia, Pseudomonas, Serratia, and Chryseobacterium showed high proteolytic activity. The highest proteolytic activeness was shown by Yersinia intermedia followed by Pseudomonas fluorescens (d). Lipolytic activeness was high in isolates of Acinetobacter, and the highest in Acinetobacter guillouiae. Certain isolates showed positive β-galactosidase and phospholipase activity. Strains belonging to the same species sometimes showed markedly different phenotypic characteristics. Proteases and lipases produced by psychrotrophic bacteria retained action afterward heat treatment at lxx, eighty, or 90 °C, and proteases appeared to exist more heat-stable than lipases. For these reasons, thermo-stable spoilage enzymes produced by a high number of psychrotrophic bacterial isolates from raw milk are of major business organization to the dairy manufacture. The results of this study provide valuable data about the spoilage potential of bacterial strains in raw milk and the thermal resistance of the enzymes they produce.
Keywords: Spoilage enzyme, Psychrotrophic bacteria, Raw milk, Thermo-stability
1. Introduction
Raw milk serves every bit an ideal medium for the growth of bacteria due to its loftier nutritional value (Champagne et al., 1994). A cold chain system is commonly used for controlling the growth of leaner in raw milk during storage and transportation. Psychrotrophic leaner are ubiquitous organisms that have the ability to grow at 7 °C or below, regardless of their higher optimal growth temperature (Sørhaug and Stepaniak, 1997). These bacteria ordinarily account for less than ten% of the full microflora of raw milk, merely invariably become predominant during the transportation and prolonged storage of raw milk at low temperatures (Sørhaug and Stepaniak, 1997).
Raw milk can be contaminated with psychrotrophic leaner from a variety of sources including air, h2o, soil, and milking equipment (Vacheyrou et al., 2011). Among psychrotrophic bacteria, genera of Pseudomonas, Acinetobacter, Flavobacterium, Chryseobacterium, and Serratia are the most frequently isolated from raw milk (Vithanage et al., 2016; Yuan et al., 2017).
Increasing global demand for dairy products requires dairy manufacturers to produce products with loftier quality and prolonged shelf-life. The dairy industry is facing constraints related to maintaining high quality and avoiding losses as a result of microbial spoilage. Mesophilic and thermophilic spore forming leaner take the potential to contaminate processed dairy products, which may then fail to comply with specifications for spore content (Sadiq et al., 2016). All the same, psychrotrophic leaner nowadays more than serious challenges to the dairy industry. Raw milk is contaminated with heat-stable enzymes produced by a broad spectrum of psychrotrophic bacteria at low temperatures, which can survive all successive processing conditions and remain active in processed dairy products (Vithanage et al., 2016). For case, the decimal reduction fourth dimension (D-value) of proteases produced past Pseudomonas spp. was 416.67 min at 75 °C and 250 min at 85 °C, indicating that these enzymes are difficult to inactivate by normal thermal processing techniques (Machado et al., 2016). Consequently, these rut-stable enzymes may lead to unacceptable biochemical changes, a decrease in nutritional value, and reduced shelf-life of dairy products (Stoeckel et al., 2016). Lipases catalyze the hydrolysis of triglycerides which cause rancid, butyric, or soapy flavors and besides may pb to a reduction in milk foaming properties (Chen et al., 2003; Bekker et al., 2016). Proteases hydrolyze casein fractions and produce defects described every bit biting off-flavors and event in age gelation (Stoeckel et al., 2016). Phospholipases degrade the integrity of the milk fat globule membrane, facilitating more lipolysis by milk'southward natural lipases (Lilbaek et al., 2007). β-Galactosidases catalyze the hydrolysis of β-ane,4-galactosidic bonds in lactose of milk (Deeth et al., 2002; Chen et al., 2009). Therefore, spoilage caused by psychrotrophic bacteria and their enzymes is a major concern in the dairy industry.
In our previous written report (Yuan et al., 2017), a various range of psychrotrophic leaner were institute in Chinese raw milk, only their ability to produce spoilage enzymes or the thermo-stability patterns of these enzymes were not studied. Thus, the objective of this study was to explore the enzymatic characteristics of all bacterial isolates in Chinese raw milk at both room temperature (28 °C) and refrigerated temperature (vii °C), and to evaluate the thermo-resistance of spoilage enzymes, thereby enhancing cognition of the spoilage potential of psychrotrophic bacteria.
2. Materials and methods
2.1. Bacterial strains
Four-hundred and eighty strains previously isolated from Chinese raw milk samples were studied (Yuan et al., 2017). These strains, representing 24 genera, 74 species, and 85 dissimilar random amplified polymorphic DNA (RAPD)-based genetic groups, were maintained in nutrient broth containing twenty% glycerol (Sinopharm Chemic Reagent Co., Ltd., Mainland china) at −80 °C.
2.ii. Screening for enzyme production
All strains were checked for their abilities to produce protease, lipase, β-galactosidase, and phospholipase on the post-obit screening media: plate count agar supplemented with 6% (0.06 g/ml) skimmed milk powder (Difco, USA) for protease; tributyrin agar (Sigma-Aldrich, United states) for lipase; nutrient agar plate spread with 35 μl v-bromo-iv-chloro-3-indolyl-β-D-galactopyranoside (Ten-gal; Sigma-Aldrich, USA) and 20 μl isopropyl-β-D-thiogalactopyranoside (IPTG; Sigma-Aldrich, Usa) for β-galactosidase; and nutrient agar plate supplemented with eight% (v/five) egg yolk emulsion (Oxoid, UK) for phospholipase. Each examination was performed at vii °C for vii d and 28 °C for 2 d.
ii.three. Quantitative assessment of total proteolytic action
Strains that showed positive protease production on screening medium were selected to quantify their full proteolytic activeness. Strains were first grown in nutrient goop for 36 h on a shaking incubator at 150 r/min. They were and then diluted in 50 ml commercial ultra-heat-treated (UHT) milk to a concluding concentration of ane×104 colony-forming unit (CFU)/ml, corresponding to the psychrotrophic bacterial cell density institute in our previous study (Yuan et al., 2017), and incubated on a shaking incubator at 120 r/min under ii different atmospheric condition: 7 °C for 7 d and 28 °C for ii d.
Samples were centrifuged at 12 000g for 10 min at iv °C afterwards the incubation. Proteolytic activity was measured using azocasein (Sigma, USA) every bit a substrate following the protocol described past Santos et al. (1996) with some modifications. To initialize the reaction, 100 μl cell-gratuitous supernatant was mixed with 500 μl phosphate buffer saline (PBS; 50 mmol/Fifty, pH seven.ii, Sangon Biotech Co., Ltd., China). And so, 100 μl azocasein (1.5% (15 one thousand/L), dissolved in 50 mmol/Fifty PBS) was added. The mixture was incubated at 37 °C for 60 min and the reaction was stopped by calculation 500 μl of 20% (0.ii g/ml) trichloroacetic acrid (Aladdin, China). After centrifugation at 12 000g for 5 min at 4 °C, absorbance at 366 nm was measured using a spectrometer-based absorbance microplate reader (Thermo Fisher Multiskan FC, Us). A control was included, in which 100 μl of sterile PBS was used in place of supernatant. The proteolytic activity is expressed as the increase of absorption at 366 nm per hour and milliliter (∆A h−1·ml−1). Each analysis was performed in triplicate and with three replicates.
2.4. Quantitative assessment of full lipolytic activeness
Strains that showed positive lipase production on screening medium were selected to quantify the total lipolytic activity. Strains were grown in nutrient broth for 36 h on a shaking incubator at 150 r/min, and then diluted in fifty ml UHT milk to a concentration of ane×teniv CFU/ml. The milk samples were and then incubated on a shaking incubator at 120 r/min under two different conditions: vii °C for 7 d and 28 °C for two d.
After incubation, samples were centrifuged at 15 000grand for xx min at 4 °C, and the supernatants were filtered using 0.45-μm cellulose acetate filter units to avoid the interference of milk proteins (Abdou, 2003; Chen et al., 2003). Lipolytic activity was measured using p-nitrophenol palmitate (p-NPP; Sigma, USA) as a substrate following the method described past Teh et al. (2013) with slight modifications. To initialize the reaction, 100 μl of cell-free supernatant was added to 900 μl substrate solution containing 3 mg of p-NPP dissolved in 1 ml isopropanol (Aladdin, China), 9 ml of l mmol/50 Tris-HCl (pH 8) solution containing 40 mg of Triton Ten-100 (Aladdin, China), and 1 mg of arabic gum (Aladdin, China). The mixture was incubated at 37 °C for 20 min and the reaction was stopped by adding 500 μl 95% ethanol (Sinopharm Chemic Reagent Co., Ltd., Prc). After centrifugation at 15 000thou for 5 min at 4 °C, absorbance at 410 nm was measured using a spectrometer-based absorbance microplate reader. A control was included, in which 100 μl sterile PBS was used in identify of the supernatant. The lipolytic activity was expressed as the amount of p-nitrophenol (μmol) released per infinitesimal per milliliter (μmol/(min·ml)) of milk sample nether the analysis conditions described higher up. Each assay was performed in triplicate and with iii replicates.
two.5. Estrus resistance of proteases and lipases
Strains that proved to be positive for protease or lipase production were selected to make up one's mind the thermo-resistance of their enzymes. Strains were grown in nutrient broth for 36 h on a shaking incubator at 150 r/min and then diluted in fifty ml UHT milk to a concentration of i×ten4 CFU/ml, followed past incubation on a shaking incubator at 120 r/min and 7 °C for vii d or 28 °C for two d. Samples were rut-treated in a h2o bath at 70 °C for 15 min. Afterwards heat treatment, samples were cooled immediately in an ice bath followed by the addition of 0.01% (0.1 g/L) sodium azide (Sinopharm Chemical Reagent Co., Ltd., China) and 0.025% (0.25 chiliad/L) bronopol (Aladdin, China) to inhibit bacterial growth (Machado et al., 2016). Subsequently, milk samples were incubated at 37 °C for 5 d. Proteolytic and lipolytic activity was measured as described above.
Strains shown to produce heat-stable enzymes in the tests described in a higher place were selected for further thermal inactivation trials. Milk samples divided into aliquots of 5 ml in sterile tubes were oestrus-treated nether the following time-temperature conditions. For protease, three different temperature-fourth dimension combinations were used: 70 °C for 180 min, 80 °C for 150 min, and 90 °C for 100 min. For lipases, dissimilar temperature-fourth dimension combinations were used: 70 °C for 80 min, 80 °C for 70 min, and 90 °C for 60 min. Afterwards each heat treatment, samples were cooled immediately in an ice bath followed by the addition of 0.01% (0.1 g/50) sodium azide and 0.025% (0.25 g/L) bronopol. Afterwards, milk samples were incubated at 37 °C for 5 d. Log of pct residue activity (R) was plotted against heating fourth dimension and expressed as the thermo-stability. The rate constant g for first-society inactivation, activation energy (E a), one-half-life (t one/2), D-value, modify in enthalpy (∆H #), change in entropy (∆S #), and change in free free energy (∆Yard #) were calculated according to Olusesan et al. (2011).
3. Results
3.1. Screening for enzyme product
A total of 423 isolates incubated at 28 °C showed the ability to produce at least one type of enzyme, while 166, 142, and 32 isolates produced two, three, and 4 unlike enzymes, respectively (Fig. 1). Isolates of Pseudomonas, Acinetobacter, Flavobacterium, Chryseobacterium, Serratia, and Aeromonas were not merely the most predominant in raw milk samples, only also proved to be high enzyme producers. Amid the Pseudomonas isolates, 235 showed proteolytic activeness and 164 showed lipolytic activeness (Fig. 2a). As well, 153 isolates showed phospholipase activity and 17 showed β-galactosidase activity. Species of Acinetobacter exhibited high lipolytic (63 out of 64 strains) only weak proteolytic (14 out of 64) and phospholipase (22 out of 64) activity, and none of them produced β-galactosidase. Of the 29 Flavobacterium isolates, 27, x, eleven, and 21 isolates showed proteolytic, lipolytic, β-galactosidase, and phospholipase action, respectively. Isolates belonging to Serratia displayed a strong trend to produce proteases (xv out of 15 strains), lipases (14 out of fifteen), and phospholipases (fifteen out of 15), but a weak ability to produce β-galactosidases (1 out of fifteen). Species belonging to Aeromonas produced all four types of enzymes, indicating their high spoilage potential. Sphingobacterium and Rahnella produced just β-galactosidases, and Janthinobacterium showed negative protease and lipase production
Number of isolates that produce different types of enzymes at seven and 28 °C
Abilities of isolates to produce protease, lipase, β-galactosidase, and phospholipase at 28 °C (a) and 7 °C (b)
The number in the bracket implies the full number of strains for each genus
Similar results were obtained at 7 °C, only slightly fewer isolates belonging to each genus showed spoilage potential. A full of 403 isolates showed the power to produce at to the lowest degree ane type of enzyme, while 143, 133, and 18 isolates produced two, three, and four different enzymes, respectively (Fig. 1). Among the Pseudomonas isolates, 221, 156, 17, and 134 isolates showed proteolytic, lipolytic, β-galactosidase, and phospholipase action (Fig. 2b), respectively. Species of Acinetobacter exhibited high lipolytic (56 out of 64 strains), but weak proteolytic (10 out of 64) and phospholipase (22 out of 64) action. Amid Flavobacterium isolates, 24, ix, 8, and 19 isolates showed proteolytic, lipolytic, β-galactosidase, and phospholipase activity, respectively. The abilities of isolates belonging to Serratia, Aeromonas, Sphingobacterium, and Rahnella to produce each enzyme were the same as those observed at 28 °C.
three.ii. Quantitative assessment of full proteolytic activity
The proteolytic activeness of the predominant psychrotrophic bacteria is shown in Fig. 3. The range of proteolysis was one.23–13.02 h−1·ml−1 at 28 °C and 0.42–5.72 h−1·ml−i at seven °C. At 28 °C, Yersinia intermedia showed the highest proteolytic activity followed by Pseudomonas fluorescens (d), Chryseobacterium oncorhynchi, Serratia grimesii, and Pseudomonas gessardii. Species of Acinetobacter showed low proteolytic action, and the everyman proteolytic activity was shown by Acinetobacter johnsonii (a). Isolates belonging to Serratia showed high proteolytic activity with a mean value of 8.51 h−1·ml−1.
Proteolytic activity of psychrotrophic bacteria isolated from raw milk
(a) Isolates belonging to Pseudomonas incubated at 28 °C; (b) Isolates belonging to other predominated species incubated at 28 °C; (c) Isolates belonging to Pseudomonas incubated at 7 °C; (d) Isolates belonging to other predominated species incubated at 7 °C. Data are expressed as mean±standard deviation (n=3)
The proteolytic activity of isolates incubated at vii °C was lower than that at 28 °C. The highest protease action was shown by Y. intermedia, followed by P. fluorescens (d), C. oncorhynchi, and Serratia liquefaciens. Rheinheimera chironomi showed the lowest protease activity followed by A. johnsonii.
iii.3. Quantitative assessment of full lipolytic activity
The lipolytic activity of the predominant psychrotrophic bacteria is shown in Fig. 4. The range of lipolytic activeness was 0.20–13.09 μmol/(min·ml) at 28 °C and 0.eleven–seven.54 μmol/(min·ml) at 7 °C. At 28 °C, Acinetobacter guillouiae showed the highest lipolytic activeness followed by Pseudomonas coleopterorum, Acinetobacter pakistanensis, and A. johnsonii (b). The everyman lipase activity was shown by Pseudomonas poae. Isolates belonging to Acinetobacter showed relatively high lipase activity with a mean value of seven.xv μmol/(min·ml).
Lipolytic activity of psychrotrophic bacteria isolated from raw milk
(a) Isolates belonging to Pseudomonas incubated at 28 °C; (b) Isolates belonging to other predominated species incubated at 28 °C; (c) Isolates belonging to Pseudomonas incubated at vii °C; (d) Isolates belonging to other predominated species incubated at 7 °C. Information are expressed every bit hateful±standard deviation (north=3)
The lipolytic activity of each isolate incubated at 7 °C was also lower than that of corresponding bacteria incubated at 28 °C. The highest lipolytic activity was shown by A. guillouiae followed by P. coleopterorum, A. johnsonii (b), and Pseudomonas extremorientalis. P. poae showed the lowest lipolytic activity followed by Flavobacterium frigidarium.
three.4. Heat resistance of proteases and lipases
Milk samples incubated at 7 °C were heat-treated at 70 °C for 15 min. As shown in Fig. v, 49 isolates showed a rest proteolytic action of <30%. The highest proportion of isolates exhibited a residual activity from 30% to 70%. Eighty-4 isolates had a residual action of ≥70%. For lipases, rest activity was <30% for 77 isolates, ≥70% for 48 isolates, and from thirty% to 70% for 137 isolates.
Distribution of isolates based on their residual action (R) of proteases (a) and lipases (b) after the oestrus handling at seventy °C for xv min
Residual action was compared later the incubation of samples at 28 °C. Fifty-four isolates showed <30%, 91 showed ≥70%, and 179 showed from 30% to 70% residual activity. For lipases, 82 isolates showed <thirty%, 54 showed ≥lxx%, and 142 showed from thirty% to 70% residue action.
Strains that showed high (≥70%) remainder proteolytic activity (P. fluorescens (d), Pseudomonas azotoformans (a), Southward. grimesii, Chryseobacterium polytrichastri, and Flavobacterium frigidimaris (b)) or lipolytic activity (A. johnsonii (b), A. pakistanensis, P. fluorescens (c), and P. azotoformans (a)) were selected for evaluating kinetic parameters of thermal inactivation. An increment in enzyme inactivation correlated well with temperature and heating time. D-values and t i/2 decreased with increasing temperature (Table 1). At vii °C, the D-values of proteases produced by these 5 isolates ranged from 116.34 to 220.51 min at 70 °C, 87.40 to 133.59 min at lxxx °C, and 59.83 to 77.13 min at 90 °C. E a ranged from 31.33 to 53.72 kJ/mol, and ∆S # from −171.23 to −111.14 J/(mol·K) at 70 °C, −164.48 to −104.29 J/(mol·G) at fourscore °C, and −157.55 to −97.65 J/(mol·K) at ninety °C. No significant differences in these kinetic parameters were observed when samples were incubated at 28 °C. Proteases produced by S. grimesii proved to be the virtually resistant, both at 28 and vii °C.
Tabular array i
Thermodynamic parameters of proteases produced by representative isolates cultured at 28 and vii °C
| Cultured temp (°C) | Isolate | Temp | 1/temp (×x−3 K−one) | 1000 (min−i) | D (min) | t 1/2 (min) | Eastward a (kJ/mol) | ∆H # (kJ/mol) | ∆S # (J/(mol·M)) | ∆One thousand # (kJ/mol) | |
| (°C) | (K) | ||||||||||
| 28 | Pseudomonas fluorescens (d) | lxx | 343 | 2.92 | 0.013 | 171.17 | 50.48 | 48.33 | 45.48 | −124.86 | 88.24 |
| eighty | 353 | 2.83 | 0.021 | 107.82 | 30.91 | 45.twoscore | −117.95 | 87.08 | |||
| 90 | 363 | 2.75 | 0.035 | 65.79 | 18.81 | 45.32 | −111.38 | 85.82 | |||
| Pseudomonas azotoformans (a) | seventy | 343 | two.92 | 0.012 | 177.80 | 52.78 | 49.44 | 46.59 | −122.23 | 88.45 | |
| 80 | 353 | two.83 | 0.021 | 111.17 | 30.71 | 46.51 | −114.81 | 87.08 | |||
| ninety | 363 | 2.75 | 0.033 | 69.xl | 18.67 | 46.43 | −108.74 | 85.97 | |||
| Serratia grimesii | lxx | 343 | two.92 | 0.010 | 225.75 | 66.66 | 53.72 | fifty.87 | −111.14 | 88.93 | |
| lxxx | 353 | 2.83 | 0.017 | 135.19 | 39.thirty | 50.79 | −104.29 | 87.64 | |||
| 90 | 363 | 2.75 | 0.030 | 77.59 | 22.03 | l.71 | −97.65 | 86.22 | |||
| Chryseobacterium polytrichastri | lxx | 343 | ii.92 | 0.019 | 122.27 | 33.66 | 33.64 | 30.79 | −164.89 | 87.26 | |
| 80 | 353 | 2.83 | 0.026 | 88.22 | 22.eighteen | 30.71 | −157.94 | 86.52 | |||
| 90 | 363 | 2.75 | 0.038 | 59.87 | fifteen.56 | xxx.63 | −151.xx | 85.61 | |||
| Flavobacterium frigidimaris (b) | lxx | 343 | 2.92 | 0.017 | 133.05 | 37.16 | 36.72 | 33.35 | −158.26 | 87.55 | |
| 80 | 353 | two.83 | 0.026 | 90.28 | 23.61 | 33.27 | −149.79 | 86.52 | |||
| ninety | 363 | 2.75 | 0.036 | 63.42 | 16.44 | 33.xix | −144.54 | 85.75 | |||
| | |||||||||||
| vii | Pseudomonas fluorescens (d) | seventy | 343 | 2.92 | 0.014 | 166.37 | 47.73 | 44.92 | 42.07 | −134.26 | 88.05 |
| eighty | 353 | two.83 | 0.022 | 106.67 | 30.59 | 41.99 | −127.24 | 86.95 | |||
| 90 | 363 | 2.75 | 0.035 | 65.21 | eighteen.23 | 41.91 | −120.75 | 85.82 | |||
| Pseudomonas azotoformans (a) | 70 | 343 | 2.92 | 0.013 | 176.21 | 51.21 | 46.xc | 44.05 | −129.03 | 88.24 | |
| eighty | 353 | 2.83 | 0.021 | 110.53 | xxx.07 | 43.97 | −122.00 | 87.08 | |||
| 90 | 363 | 2.75 | 0.034 | 68.41 | eighteen.05 | 43.89 | −115.53 | 85.xc | |||
| Serratia grimesii | 70 | 343 | ii.92 | 0.010 | 220.51 | 58.46 | 53.72 | l.87 | −111.xiv | 88.93 | |
| 80 | 353 | 2.83 | 0.017 | 133.59 | 37.70 | fifty.79 | −104.29 | 87.64 | |||
| 90 | 363 | 2.75 | 0.030 | 77.13 | 21.58 | 50.71 | −97.65 | 86.22 | |||
| Chryseobacterium polytrichastri | 70 | 343 | ii.92 | 0.020 | 116.34 | 32.00 | 31.33 | 28.48 | −171.23 | 87.12 | |
| 80 | 353 | 2.83 | 0.026 | 87.forty | 21.36 | 28.twoscore | −164.48 | 86.52 | |||
| 90 | 363 | 2.75 | 0.038 | 59.83 | 15.24 | 28.32 | −157.55 | 85.61 | |||
| Flavobacterium frigidimaris (b) | 70 | 343 | 2.92 | 0.018 | 130.05 | 35.46 | 34.23 | 31.38 | −163.58 | 87.40 | |
| eighty | 353 | 2.83 | 0.026 | 90.02 | 22.71 | 31.xxx | −156.27 | 86.52 | |||
| 90 | 363 | two.75 | 0.036 | 63.53 | 16.24 | 31.22 | −149.96 | 85.75 | |||
Lipases showed less rut resistance than proteases (Tabular array 2). At 7 °C, D-values of lipases produced past the four isolates ranged from 44.31 to 72.74 min at seventy °C, 35.41 to 57.44 min at 80 °C, and 28.98 to 40.69 min at 90 °C. E a ranged from 21.03 to 28.20 kJ/mol, and ∆Due south # from −194.04 to −176.81 J/(mol·M) at 70 °C, −186.89 to −170.17 J/(mol·Yard) at 80 °C, and −180.62 to −163.27 J/(mol·Chiliad) at ninety °C. Similarly, no significant differences in these kinetic parameters were observed when samples were incubated at 28 °C. Lipase produced by A. pakistanensis showed the highest heat resistance, both at 28 and vii °C.
Tabular array 2
Thermodynamic parameters of lipases produced by representative isolates cultured at 28 and seven °C
| Cultured temp (°C) | Isolate | Temp | 1/temp (×x−iii K−1) | chiliad (min−1) | D (min) | t i/two (min) | E a (kJ/mol) | ∆H # (kJ/mol) | ∆S # (J/(mol·K)) | ∆G # (kJ/mol) | |
| (°C) | (K) | ||||||||||
| 28 | Acinetobacter johnsonii (b) | seventy | 343 | 2.92 | 0.033 | 70.17 | 21.56 | 27.24 | 24.39 | −179.38 | 85.82 |
| fourscore | 353 | 2.83 | 0.041 | 56.64 | xv.46 | 24.31 | −172.69 | 85.33 | |||
| xc | 363 | 2.75 | 0.058 | 39.41 | ten.60 | 24.23 | −165.77 | 84.51 | |||
| Acinetobacter pakistanensis | seventy | 343 | two.92 | 0.031 | 75.43 | 23.96 | 28.73 | 25.45 | −176.75 | 85.98 | |
| eighty | 353 | ii.83 | 0.040 | 58.23 | 16.32 | 25.37 | −169.88 | 85.40 | |||
| 90 | 363 | 2.75 | 0.056 | 41.06 | 11.forty | 25.29 | −163.ten | 84.60 | |||
| Pseudomonas fluorescens (c) | seventy | 343 | 2.92 | 0.051 | 45.55 | 13.44 | 21.51 | 18.66 | −192.81 | 84.69 | |
| 80 | 353 | 2.83 | 0.064 | 36.xiii | 10.ten | xviii.58 | −185.65 | 84.18 | |||
| 90 | 363 | 2.75 | 0.079 | 29.07 | 8.xi | 18.50 | −169.62 | 83.74 | |||
| Pseudomonas azotoformans (a) | 70 | 343 | ii.92 | 0.041 | 56.42 | sixteen.87 | 25.94 | 23.09 | −181.51 | 85.25 | |
| fourscore | 353 | 2.83 | 0.055 | 41.59 | 11.67 | 23.01 | −174.21 | 84.57 | |||
| 90 | 363 | 2.75 | 0.070 | 32.74 | viii.60 | 22.94 | −167.97 | 84.02 | |||
| | |||||||||||
| 7 | Acinetobacter johnsonii (b) | lxx | 343 | 2.92 | 0.033 | 69.92 | 21.31 | 27.24 | 24.39 | −179.38 | 85.82 |
| fourscore | 353 | two.83 | 0.041 | 56.13 | xv.20 | 24.31 | −172.69 | 85.33 | |||
| ninety | 363 | 2.75 | 0.058 | 39.55 | 10.62 | 24.23 | −165.77 | 84.51 | |||
| Acinetobacter pakistanensis | lxx | 343 | two.92 | 0.032 | 72.74 | 21.66 | 28.20 | 25.35 | −176.81 | 85.90 | |
| 80 | 353 | 2.83 | 0.040 | 57.44 | 15.77 | 25.27 | −170.17 | 85.40 | |||
| xc | 363 | 2.75 | 0.057 | 40.69 | 11.28 | 25.xix | −163.27 | 84.56 | |||
| Pseudomonas fluorescens (c) | 70 | 343 | 2.92 | 0.052 | 44.31 | 12.78 | 21.03 | 18.eighteen | −194.06 | 84.64 | |
| 80 | 353 | two.83 | 0.065 | 35.41 | 9.67 | xviii.10 | −186.89 | 84.xiv | |||
| 90 | 363 | two.75 | 0.079 | 28.98 | seven.77 | 18.02 | −180.62 | 83.70 | |||
| Pseudomonas azotoformans (a) | 70 | 343 | 2.92 | 0.042 | 55.34 | 16.23 | 25.46 | 22.61 | −182.73 | 85.19 | |
| 80 | 353 | 2.83 | 0.056 | 41.nineteen | 11.53 | 22.53 | −175.43 | 84.52 | |||
| 90 | 363 | 2.75 | 0.071 | 32.60 | eight.47 | 22.45 | −169.21 | 83.98 | |||
4. Discussion
Heat-stable enzymes produced by psychrotrophic leaner cannot be completely inactivated by thermal processing techniques and pose threats to the quality and shelf-life of dairy products. Some studies accept reported results of screening for enzymes produced by psychrotrophic bacteria isolated from raw milk (von Neubeck et al., 2015; Vithanage et al., 2016). However, quantitative assessments and the thermo-stabilities of proteases and lipases have rarely been reported. The objective of this work was to explore the spoilage potential of psychrotrophic leaner and the thermo-stabilities of proteases and lipases they produced.
A wide variety of psychrotrophic leaner tin produce spoilage enzymes during tardily log or stationary growth phases (Rajmohan et al., 2002). Near isolates in this written report had enzymatic activity at low temperatures. Pseudomonas is reported to exist the nigh frequently isolated genus in raw milk, probably because of its short generation fourth dimension (von Neubeck et al., 2015; Yuan et al., 2017). The spoilage potential of Pseudomonas has been extensively studied and those bacteria tend to produce proteases combined with lipases (Capodifoglio et al., 2016; Vithanage et al., 2016).
In this study, about Pseudomonas species, such as P. fluorescens (strains b and d), P. gessardii, P. lurida, and P. brenneri, showed high protease activity, while P. azotoformans (a), P. coleopterorum, P. extremorientalis, P. fragi (c), P. fluorescens (c), and P. lurida showed loftier lipase activeness. P. fluorescens, P. lurida, and P. simiae can produce all 4 of the different enzymes screened in this work, indicating their swell spoilage potential. The aprX gene coding for the protease is located at the beginning of a polycistronic operon with the lipA gene that encodes the lipase (Woods et al., 2001). Thus, virtually isolates belonging to to Pseudomonas have the ability to produce proteases and lipases due to the simultaneous expression of both genes. Nonetheless, expression of enzymes tin exist tightly regulated, which may explain the absenteeism of one or both enzymes in some isolates. In this work, some isolates belonging to P. fluorescens and P. fragi
did not show proteolytic or lipolytic action. This phenotypic variation within the aforementioned species is consistent with previous findings (Wiedmann et al., 2000; Caldera et al., 2016). For example, but 36% of isolates belonging to P. fragi from different foods showed proteolytic activity (Caldera et al., 2016). For this reason, in the future, whole genome sequencing of these isolates will be carried out to differentiate their spoilage potential.
Acinetobacter is considered to be the predominant bacterial genus in raw milk (von Neubeck et al., 2015; Yuan et al., 2017). This genus is ubiquitous in nature and characterized by the tendency to tolerate dry conditions and multidrugs (Gurung et al., 2013). In the current work, all isolates belonging to Acinetoba showed high lipolytic activity and low proteolytic action, which is in understanding with previous studies (von Neubeck et al., 2015; Vithanage et al., 2016). However, a high number of Acinetobacter isolates with proteolytic action were found in Mozzarella cheese. Such isolates tin carry out the hydrolysis of α-casein and spoil the cheese (Baruzzi et al., 2012). This might exist explained by horizontal gene transfer from proteolytic species or phenotypic variation among isolates. Chryseobacterium also appears every bit a ascendant fellow member of raw milk microbiota (Yuan et al., 2017) and some species evidence a greater spoilage ability than P. fluorescens based on their proteolytic and lipolytic activity (Bekker et al., 2015, 2016). In this study, species of Chryseobacterium showed more than proteolytic than lipolytic activity, and C. oncorhynchi showed higher proteolytic activity than many isolates of Pseudomonas. To our knowledge, few studies accept reported the spoilage potential of Flavobacterium. The results of our study propose that it has high proteolytic but weak lipolytic activity. Species of Serrati are also characterized every bit predominant milk spoilers due to their power to produce heat-stable proteases and lipases (Machado et al., 2016). In this written report, Serratia was shown to have loftier spoilage potential based on its ability to produce protease, lipase, β-galactosidase, and phospholipase. However, isolates belonging to other predominant genera (Sphingobacterium, Rahnella, Buttiauxella, and Janthinobacterium) lacked the ability to produce both proteases and lipases. Thus, the population of psychrotrophic bacteria plays a key role in the determination of the quality of dairy products.
The productions of phospholipases and β-galactosidases have been less frequently reported than those of proteases and lipases, since proteases and lipases pose more serious threats to the quality and shelf-life of dairy products. Phospholipase activity was institute amid the isolates belonging to the genera Pseudomonas, Acinetobacter, Flavobacterium, Serratia, Chryseobacterium, Janthinobacterium, and Aeromonas, consequent with their ability to cause sweet curdling defects or biting cream in milk as a consequence of fatty globule aggregation (Titball, 1993; Vithanage et al., 2016). β-Galactosidase is an of import indicator of lactose fermentation in dairy products, and may catalyze the hydrolysis of β-1,iv-galactosidic bonds in lactose (Chen et al., 2009). The product of β-galactosidase by psychrotrophic leaner has seldom been reported. In this study, β-galactosidases were produced by isolates of the genera Pseudomonas, Flavobacterium, Sphingobacterium, Rahnella, Buttiauxella, Rheinheimera, Sphingobacterium, and Aeromonas.
The production of enzymes is a temperature-dependent process (Buchon et al., 2000). As reported by Decimo et al. (2014), enzymatic activeness is highly influenced by the incubation temperature as results obtained at 30 °C differ from those obtained at vii °C. In our piece of work, more than isolates showed positive results for each enzymatic activity, and activity of proteases and lipases was college at 28 °C than at seven °C, indicating that the storage and transportation of raw milk at low temperatures could be constructive to some extent in decision-making the spoilage of raw milk.
Considering the potential effects of enzymes on dairy products, appropriate practices should be adopted to minimize the quality losses, by either limiting the growth of psychrotrophic bacteria or inactivation of their enzymes by thermal processing. Acceptable addition of CO2 to raw milk has been shown to reduce proteolysis and lipolysis by limiting microbial growth and the product of microbial proteases (Ma et al., 2003). Sophisticated dairy subcontract management systems, such as Proficient Manufacturing Practice (GMP) and Hazard Analysis and Critical Control Point (HACCP), are also effective ways to limit the initial number of psychrotrophic bacteria in raw milk (Cusato et al., 2014). Withal, the effects of these practices on the heat stability of enzymes are unknown. The heat stability of enzymes originating from psychrotrophs is a limiting factor in maintaining the quality and shelf-life of dairy products. Thus, ameliorate knowledge of their heat stability is needed.
Heat treatments adopted by the dairy manufacture are insufficient to completely inhibit the activity of enzymes produced past psychrotrophic leaner (Glück et al., 2016; Vithanage et al., 2016). Reducing the action and limiting the secretion of estrus-stable enzymes are scientific challenges. In this work, proteases produced past a large number of predominant isolates still remained agile after oestrus handling at 70 °C for fifteen min, and some fifty-fifty showed residual activity of ≥70%. Kinetic parameters for the thermal inactivation of P. fluorescens (d), P. azotoformans (a), and Serratia quinivorans highlight the high potential of these enzymes to cause dairy spoilage. In previous studies, the thermo-stability of proteases produced by Pseudomonas or Serratia has been extensively reported. The D-values of proteases produced by S. liquefaciens and Pseudomonas sp. were 96.xv and 80.00 min, respectively, at ninety °C (Machado et al., 2016). The D-values of proteases produced by Pseudomonas proteolytica were 6.3, four.0, and 1.3 min at 120, 130, and 140 °C, respectively (Stoeckel et al., 2016). The remainder activity of protease produced by P. fluorescens was 97.2% after oestrus treatment at 80 °C for xxx min (Zhang and Lv, 2014). The purified protease Ser2 secreted past S. liquefaciens was highly heat-stable in skimmed, semi-skimmed, and whole milk at 140 °C, with D-values of two.four, 3.9, and 4.5 min, respectively, and the presence of milk fatty increased the heat-stability of the protease (Baglinière et al., 2017). The estrus inactivation of the purified protease from Chryseobacterium indologenes did not follow first-order kinetics, just showed biphasic inactivation curves, and this protease is much more than oestrus-labile than other psychrotrophic proteases (Venter et al., 1999). In our study, the kinetic parameters for inactivation of the protease secreted by C. polytrichastri suggested that this protease is also heat-resistant. This may exist because the crude enzyme is more heat-stable than the purified enzyme, and the unlike methods for estimating oestrus resistance should besides exist taken into business relationship. Kinetic thermal inactivation of proteases produced by species belonging to Flavobacterium has not been reported previously, and our results suggest that the protease produced past F. frigidimaris (b) is thermo-stable.
Many studies have determined the heat stability of proteases, but the oestrus stability of lipases is less well known. Information technology has been reported that lipases produced by diverse Pseudomonas isolates showed rest activity ranging from 55% to 100% after rut handling at 63 °C for 30 min (Law et al., 1976). The D-value of lipase produced by P. fluorescens was 23.5 min later on heat handling at 100 °C (Andersson et al., 1979). Vithanage et al. (2016) reported that more than than 30% of strains belonging to Pseudomonas isolated from raw milk showed fifty% to 75% residual lipase activity afterward estrus treatment at 142 °C for 4 s. In this study, many isolates showed high residue lipase activity afterwards heat handling at 70 °C for 15 min. Kinetic parameters showed that lipases produced by A. johnsonii (b), A. pakistanensis, P. fluorescens (c), and P. azotoformans (a) are heat-resistant and can survive after pasteurization. Surprisingly, lipases were less estrus-resistant than proteases. For instance, the D-value of the most heat-stable lipase produced past A. pakistanensis was 41.06 min at 90 °C, which is much smaller than that of the most heat-stable protease produced past Due south. grimesii. Moreover, enzymes are more estrus-stable in synthetic milk salt solutions than in phosphate buffer due to the protective result of milk components in the milk system (Baur et al., 2015).
The inactivation of enzymes fits first-order kinetics, and kinetic parameters indicate that while a college temperature and longer heat treatment menses may result in a higher reduction of enzymatic activity, the destruction and inactivation of milk constituents will be increased. Results for the Gibbs free energy (Thou #), enthalpy (H #), and entropy (S #) of thermal inactivation of enzymes have rarely been reported. Hydrophobic interactions, hydrogen bonds, disulfide bridges, and electrostatic interactions are the forces that stabilize a protein and are determined by ∆One thousand #, while ∆H # and ∆S # provide a measure of structural disorder upon protein folding associated with the stability of an enzyme. Loftier levels of ∆K # and ∆H #, and low values of ∆Southward # indicate the structural stability of enzymes in this written report. It is hard to compare the results of estrus stability of enzymes provided by dissimilar laboratories due to different fourth dimension-temperature combinations of estrus treatments and because heat resistance varies at the species or strain level. The thermo-stability patterns exhibited by proteases and lipases demonstrate the importance of exploring the combination of optimal temperature and time for heat treatments in the dairy industry.
The common cold-chain of storage and transportation reduces the production and activity of spoilage enzymes. However, no meaning departure in thermo-stability was observed between low and high incubation temperatures in this study. This can be explained by the fact that the thermo-stability of enzymes depends on the genetics of the specific isolates tested.
Information technology is of import to stress that this piece of work focused only on enzymes produced by planktonic cells. The amount of proteolysis and lipolysis produced is higher in biofilms than in planktonic cultures, and the thermo-stability of enzymes tin be enhanced by the protective matrix of EPS (Teh et al., 2012, 2013). This suggests that biofilms formed by spoilage psychrotrophs may pose more serious threats to the dairy manufacture. To our cognition, few studies have reported whatever possible link betwixt spoilage enzymes and biofilm formation by psychrotrophic leaner. This volition be taken into consideration in the hereafter inquiry.
five. Conclusions
The production of rut-resistant spoilage enzymes presents a formidable claiming to today's dairy manufacture. A high number of predominant bacteria isolated from Chinese dairy milk can produce spoilage enzymes. Species of Pseudomonas, Serratia, and Chryseobacterium are highly proteolytic, while isolates of Acinetobacter and Pseudomonas tin produce highly active lipases. Isolates belonging to sure genera bear witness positive β-galactosidase and phospholipase action. Almost enzymes are heat-resistant every bit they can survive after different heat treatments applied in the manufacture of dairy products. There is a vast amount of scientific knowledge nigh the ability of psychrotrophic leaner to produce spoilage enzymes, but no effective strategy has nevertheless been presented to control this trouble. Thus, the heat stability and molecular characteristics of enzymes and the development of novel screening methods should be addressed in the futurity.
Footnotes
*Projection supported by the National Natural Scientific discipline Foundation of China (No. 31772080) and the Major Science and Technology Projects of Zhejiang Province (No. 2015C02039), Mainland china
Compliance with ideals guidelines: Lei YUAN, Faizan A. SADIQ, Tong-jie LIU, Yang LI, Jing-si GU, Huan-yi YANG, and Guo-qing HE declare that they have no conflict of involvement.
This commodity does not contain whatever studies with human or creature subjects performed by whatsoever of the authors.
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