Article

Inactivation of thermoduric aerobic sporeformers in milk by ultrasonication

Authors:
To read the full-text of this research, you can request a copy directly from the authors.

Abstract

Thermoduric sporeformers can survive milk pasteurization and cause spoilages of dairy products. In the present study, ultrasonication was evaluated as a non-thermal processing technique to inactivate the thermally resistant vegetative cells of spore forming Bacillus spp. During the challenge studies, vegetative cells of Bacillus coagulans (ATCC (R) 12245), Anoxybacillus flavithermus (DSM 2641), Bacillus sporothermodurans (DSM 10599), Bacillus licheniformis (ATCC (R) 6634), and Geobacillus stearothermophilus (ATCC (R) 15952) were studied for their survivability to batch pasteurization (63 degrees C/30 min) in skim and whole milk samples. Experiments were conducted to study the effect of ultrasonication alone or in combination with pasteurization on inactivation of vegetative cells of some thermoduric Bacillus spp. Effect of ultrasonication on milk pH, color, and alkaline phosphatase activity was also investigated. Vegetative cells of B. coagulans and A. flavithermus survived pasteurization treatment in both skim and whole milk samples. Ultrasonication at 80% amplitude for 10 min however, inactivated the vegetative cells of B. coagulans and A. flavithermus in skim milk by 4.53, and 4.26 logs, respectively. A combined treatment of pasteurization (63 degrees C/30 min) followed by ultrasonication completely eliminated approximately log 6 cfu/mL of these cells in skim milk. As visualized under the scanning electron microscope, ultrasonication physically disintegrated vegetative cells of sporeformers. Ultrasonication treatment caused significant reduction (P < 0.05) in brightness and greenness of milk; whereas, blueness (b*) of milk was increased. However, pH and alkaline phosphatase activity (P > 0.05) of treated skim milk were not affected.

No full-text available

Request Full-text Paper PDF

To read the full-text of this research,
you can request a copy directly from the authors.

... The high-intensity ultrasound (HIUS) is mainly studied for processing of liquid foods, like fruit and vegetable juices and dairy products, in which induces mechanical, chemical and biochemical effects via the production and subsequent collapse of cavitation bubbles, generating energy, responsible for the physicochemical and microbiological alterations observed during food processing [4]. However, the recent studies has been directed to the application of thermosonication, which combines the application of heat with ultrasound, improving greatly the microorganisms lethality, once the application of ultrasound alone appears to induce low microbial inactivation at low temperatures [5][6][7]. In this way, the ultrasound as a non-thermal technology, without the external application of heat, is not a well-explored area of dairy products, once only few studies are found with no external heat application [8][9][10] or controlled low temperature [6,[11][12][13]. ...
... However, the recent studies has been directed to the application of thermosonication, which combines the application of heat with ultrasound, improving greatly the microorganisms lethality, once the application of ultrasound alone appears to induce low microbial inactivation at low temperatures [5][6][7]. In this way, the ultrasound as a non-thermal technology, without the external application of heat, is not a well-explored area of dairy products, once only few studies are found with no external heat application [8][9][10] or controlled low temperature [6,[11][12][13]. In contrast, increased temperatures and ultrasonic intensities for milk processing may cause sensorial deterioration and physical-chemical degradation [10,14]. ...
... Moreover, the whey beverage may contain bacterial spores, since most of the ingredient are powders (inulin, gellan gum, whey powder) [39,40]. The ultrasound and the thermal treatment may be acting as a stress factor, germinating the spores and increasing the AMHB counts, or not affecting the thermo-resistant bacteria [6], which remain in the beverage. Therefore, the different microbial composition of the pasteurized milk batches used as ingredient may be interfering in the microbial inactivation rate, once only the more sensible bacteria may have been inactivated. ...
Article
In this work, we investigated the effects of the ultrasonic power (0, 200, 400 and 600 W) on non-thermal processing of an inulin-enriched whey beverage. We studied the effects of high-intensity ultrasound (HIUS) on microbial inactivation (aerobic mesophilic heterotrophic bacteria (AMHB), total and thermotolerant coliforms and yeasts and molds), zeta potential, microstructure (optical microscopy, particle size distribution), rheology, kinetic stability and color. The non-thermal processing applying 600 W of ultrasonic power was comparable to high-temperature short-time (HTST) treatment (75 °C for 15 s) concerning the inactivation of AMHB and yeasts and molds (2 vs 2 log and 0.2 vs 0.4 log, respectively), although HIUS has reached a lower output temperature (53 ± 3 °C). The HIUS was better than HTST to improve beverage kinetic stability, avoiding phase separation, which was mainly attributed to the decrease of particles size, denaturation of whey proteins and gelation of polysaccharides (inulin and gellan gum). Thus, non-thermal processing by HIUS seems to be an interesting technology for prebiotic dairy beverages production.
... Vegetative cells and spores of thermophilic bacteria are capable of surviving pasteurisation temperatures. Thermophilic bacteria grow in dairy products and produce acids and enzymes which deteriorate milk quality, and are a concern for the dairy industry (Burgess et al. 2010;L€ ucking et al. 2013;Khanal et al. 2014b). Thermophilic bacteria have been known to cause defects such as ropiness, flat sour spoilage, and production of lactic acid and off flavours in milk products such as ultra-high-temperature (UHT) pasteurised milk and canned milk (Burgess et al. 2010;L€ ucking et al. 2013;Khanal et al. 2014b). ...
... Thermophilic bacteria grow in dairy products and produce acids and enzymes which deteriorate milk quality, and are a concern for the dairy industry (Burgess et al. 2010;L€ ucking et al. 2013;Khanal et al. 2014b). Thermophilic bacteria have been known to cause defects such as ropiness, flat sour spoilage, and production of lactic acid and off flavours in milk products such as ultra-high-temperature (UHT) pasteurised milk and canned milk (Burgess et al. 2010;L€ ucking et al. 2013;Khanal et al. 2014b). Vegetative cells and spores of thermophilic bacteria are able to form heat-resistant biofilms on equipment surfaces and can contaminate the product stream resulting in products with spore counts 1000 times higher than raw incoming milk (Burgess et al. 2010). ...
... Thermosonication and sonication combined with pressure (manosonication) or both (manothermosonication) have been effective in reducing microorganisms in foods (Villamiel et al. 2009). Sonication has been explored to improve milk quality by reducing the indigenous microbiota or added pathogens in batch and continuous systems (Berm udez-Aguirre et al. 2009a, 2009bKhanal et al. 2014aKhanal et al. , 2014b. Lim et al. (2019) studied the effect of batch thermosonication and cold sonication on milk inoculated with Paenibacillus amolyticus which is a thermotolerant psychrophilic organism. ...
Article
Thermosonication may help reduce bacteria counts responsible for spoilage in dairy products. Vegetative cells and spores of Geobacillus stearothermophilus, Anoxybacillus flavithermus and Bacillus subtilis (spores only) were treated with either heat alone or thermosonication in a batch system from 0 to 120 s in tryptic soy broth and 2% fat milk at 72 and 73 °C. D‐values for vegetative cells were calculated and were reduced after thermosonication. Maximum reduction in vegetative cells after thermosonication was 1 log after 30–45 s and for spores was ≤0.2 log after 120 s, which may not influence dairy product quality in scale‐up systems.
... • Ultrasound • Stand-alone ultrasound technology leads to lower microbial inactivation while compared to thermal pasteurization (Khanal et al., 2014;Guimarães et al., 2018) • Low temperatures and mild ultrasonic intensities may be beneficial to stimulate the growth and activity of microorganisms (Pagnossa et al., 2020) • Causes lipid oxidation in milk (Marchesini et al., 2015) • Intact protein and free amino acid profiles in semi-skimmed sheep's milk (Balthazar et al., 2019) • Higher nutritional value due to the higher production of bioactive peptides in fermented milk (Huang et al., 2019) • Longer processing times generates off-flavours (Marchesini et al., 2015) • Better parameters associated with colour and stability during product storage • Wave frequency, applied power, processing time and food temperature ...
... The high intensity ultrasound (HIUS) is mainly studied for processing of dairy products. Ultrasounds as a standalone technology are not effective in reducing microorganism counts at low temperatures, thus the use of ultrasounds as a nonthermal technology is not a well-studied area of dairy products (Khanal et al., 2014;Guimarães et al., 2018). There are limited studies on application of ultrasounds with no heat treatment (Marchesini et al., 2015) or with controlled low temperature (Gao et al., 2014;Khanal et al., 2014). ...
... Ultrasounds as a standalone technology are not effective in reducing microorganism counts at low temperatures, thus the use of ultrasounds as a nonthermal technology is not a well-studied area of dairy products (Khanal et al., 2014;Guimarães et al., 2018). There are limited studies on application of ultrasounds with no heat treatment (Marchesini et al., 2015) or with controlled low temperature (Gao et al., 2014;Khanal et al., 2014). Increased temperatures and ultrasonic intensities for milk processing may cause changes in sensory and physicochemical properties (Marchesini et al., 2015;Guimarães et al., 2018). ...
Article
Full-text available
Thermal treatment has always been the processing method of choice for food treatment in order to make it safe for consumption and to extend its shelf life. Over the past years non-thermal processing technologies are gaining momentum and they have been utilized especially as technological advancements have made upscaling and continuous treatment possible. Additionally, non-thermal treatments are usually environmentally friendly and energy-efficient, hence sustainable. On the other hand, challenges exist; initial cost of some non-thermal processes is high, the microbial inactivation needs to be continuously assessed and verified, application to both to solid and liquid foods is not always available, some organoleptic characteristics might be affected. The combination of thermal and non-thermal processing methods that will produce safe foods with minimal effect on nutrients and quality characteristics, while improving the environmental/energy fingerprint might be more plausible.
... Pasteurisation did not inactivate the vegetative cells of B. coagulans and Anoxybacillus (A.) flavithermus but B. licheniformis, B. sporothermodurans and Geobacillus (G.). stearothermophilus were not resistant (Khanal, Anand, Muthukumarappan, & Huegli, 2014a). The samples were US-treated (20 kHz, 91.2 mm, 1, 5, 10 min). ...
... Deshpande and Walsh (2021) did not, however, obtain a significant reduction in cell number in a culture of G. stearothermophilus in milk when they combined US-treatment (72 C, 7.1 s), a practical applicable time, with heat-treatment (72 C, 13.2 s). When Khanal, Anand, and Muthukumarappan (2014b) treated spores of B. coagulans, B. licheniformis and G. stearothermophilus in skimmed milk under the same US conditions as described above (Khanal et al., 2014a), the number of spores of the three bacteria were reduced by 49, 35 and 33% respectively after 10 min. US combined with pasteurisation reduced the number of spores by 66% for all three types of bacteria. ...
... Results obtained at 61 C can be of particular interest as this is close to the temperature of milk as it leaves the separator. Lack of inactivation of ALP and lactoperoxidase in milk by US-treatment at 24e26 C (20 kHz, 750 W, 124 mm) was also shown by Cameron et al. (2009), and no significant reduction in ALP activity after US-treatment for 1, 5 and 10 min was registered by Khanal et al. (2014a). The effect of US on ALP in milk was also investigated by Pegu and Arya (2021) who concluded that US for 8 min at 400 W did not inactivate ALP when the temperature in the milk sample was increased from 30 C to 52 C. Shorter processing time and lower intensity surprisingly indicated a slight increase in ALP-activity. ...
Article
Full-text available
Ultrasound (US) treatment of milk is one of several emerging technologies, often promoted as non-thermal, although a combination with heat appears to show greater potential. Bacteriocidal effects have been studied, as well as certain interesting chemical and physical changes occurring as a result of this treatment. Comparison of published results is confounded by the lack of standardised treatment parameters. US does not appear to offer advantages regarding microbiological quality, in comparison with conventional HTST pasteurisation (72 °C for 15 s). Most publications do not address possible sensory changes caused by US treatment but, according to some authors, some definitive changes do occur. To obtain knowledge that ensures that quality products are produced, research on US is needed that combines the aspects of the effect on microorganisms, product functionality and quality and the influence on flavour and sensory aspects of the milk or milk product.
... Thermophilic bacteria, yeasts, and spores can survive the current pasteurization of fruit juices and milk [17,18]. The presence of these microbes reduces product quality and shelf life due to production of acid, lipases, proteases, and offflavors during storage [17,19]. Additionally, the germination of spores is a concern in nonfat dry milk and skim milk powders because the powders themselves or food products formulated with them can have inferior quality and acceptability [17]. ...
... Higher heat treatments such as ultrahigh temperature heating improve microbial quality at the expense of product freshness. Coupling ultrasound with pasteurization may aid in reducing the thermophilic microbes in fluid foods [19] extending the quality and shelf life of the products. Previous research has shown that no off-flavors were generated in 10% whey suspensions sonicated for 15 min using a power level of 3 W; therefore product freshness should be retained in solicited milk [20]. ...
... Studies on the inactivation of yeast in juices with sonication have shown a 7-log reduction of S. cerevisiae in grape juice at 60 ∘ C [7] and greater than a 5-log reduction of Pichia fermentans in tomato juice at 40 ∘ C [9]. Temperatures greater than 40 ∘ C were necessary for the inactivation of S. cerevisiae and the maximum inactivation of S. cerevisiae was observed at 60 ∘ C. Valero et al. [26] showed a 0.6-log reduction of indigenous yeast in orange juice at 88 ∘ C yet the conditions used by Khanal et al. [19] had no effect on indigenous yeasts (10 ∘ C, 8 min, and 89 m). Our results are in agreement in that temperatures greater than 40 ∘ C are needed for the inactivation of S. cerevisiae. ...
Article
Full-text available
This study investigated the effects of high intensity ultrasound (temperature, amplitude, and time) on the inactivation of indigenous bacteria in pasteurized milk, Bacillus atrophaeus spores inoculated into sterile milk, and Saccharomyces cerevisiae inoculated into sterile orange juice using response surface methodology. The variables investigated were sonication temperature (range from 0 to 84°C), amplitude (range from 0 to 216 μm), and time (range from 0.17 to 5 min) on the response, log microbe reduction. Data were analyzed by statistical analysis system software and three models were developed, each for bacteria, spore, and yeast reduction. Regression analysis identified sonication temperature and amplitude to be significant variables on microbe reduction. Optimization of the inactivation of microbes was found to be at 84.8°C, 216 μm amplitude, and 5.8 min. In addition, the predicted log reductions of microbes at common processing conditions (72°C for 20 sec) using 216 μm amplitude were computed. The experimental responses for bacteria, spore, and yeast reductions fell within the predicted levels, confirming the accuracy of the models.
... Although ultrasound has great potential for application in the dairy industry, the technique is not currently used for processing or preservation purposes because of the limited knowledge of the effects upon shelf-limiting enzymes, sensory and other quality parameters, energy consumption, and scale-up potential. A few reports (Villamiel and de Jong, 2000a,b;Cameron et al., 2008Cameron et al., , 2009Chouliara et al., 2010;Khanal et al., 2014) have provided insight into the promise of thermosonication (Ts) for microbiological, chemical, and sensory quality of skim milk or full-fat milk, but most utilized previously pasteurized milk or conducted treatments in ice baths, and analyses were limited to milk stored for less than 1 wk. The present work was designed to evaluate skim milk and cream quality for 30 d. ...
... Differences beyond d 1, although significant, would not likely be notable by consumers, implying that the TS treatment conditions selected for this study would not adversely affect the appearance of skim milk or cream. Our results are in line with Khanal et al. (2014), who recently reported no significant changes in L* value of milk after 1 or 5 min of ultrasonication. ...
... Earlier work by Cameron et al. (2008Cameron et al. ( , 2009 reported that ultrasound (20 kHz, 750 W, 24 to 26°C, in ice bath, for 6 to 10 min) completely eliminated coliforms and yielded ≥99% reduction of pathogens (Escherichia coli; Listeria monocytogenes) and spoilage microorganisms (Pseudomonas fluorescens) inoculated into sterile whole milk (3.4% fat). More recently, sterile skim milk was inoculated with vegetative cells of spoilage microorganisms Bacillus coagulans and Anoxybacillus flavithermus (Khanal et al., 2014). When 10 min of sonication (80% amplitude, 114 μm maximum, in an ice bath) was followed by pasteurization, the treatment resulted in complete inactivation of cells; inactivation was incomplete with sonication alone and at lower sonication times (1 or 5 min) followed by pasteurization. ...
Article
Full-text available
The shelf life of pasteurized milk is limited by heat-stable proteases, which cause gelation and bitter taste upon extended storage of milk. Ultra-high temperature processing inactivates proteases but detrimentally affects milk's sensory quality. An adjunct to pasteurization is desired to extend milk's shelf life while maintaining quality characteristics. In this study, the effects of combined heat and ultrasound (thermosonication) on total plasmin activity and various quality characteristics of skim milk and cream were studied. Thermosonication, at an average power of 115 W (152 μmp-p, where p-p = peak-to-peak amplitude) for 3 min, decreased the total plasmin activity (i.e., plasmin- and plasminogen-derived activity) by nearly 94% in fresh skim milk and cream. Enzyme activity in thermosonicated skim milk samples measured at the end of 30 d was 5- to 10-fold higher than on d 0, but remained stable in thermosonicated cream. Descriptive analysis of odor attributes was conducted for up to 4 wk with 8 trained panelists. No significant differences were observed between the intensities of offensive "eggy" and "rubbery" odor attributes of thermosonicated skim milk and pasteurized commercial skim milk and cream. In addition, lightness (L*) values and viscosity were not adversely affected by thermosonication. Thermosonication decreased the fat globule size in skim milk and cream, and the homogenizing effect increased with treatment time. Thermosonication at average powers of 104 W (133 μmp-p) and 115 W (152 μmp-p) for 1 and 3 min destroyed coliforms and over 99% of the total aerobic bacteria. The total aerobic bacteria counts of thermosonicated skim milk and cream samples were less than 20,000 cfu/mL on d 30. Because thermosonication did not induce off-aromas or viscosity changes but did inactivate microorganisms and protease enzymes, thermosonication may be an appropriate adjunct to pasteurization to extend milk shelf life.
... Some preliminary studies have been performed, including a report that ultrasound treatment at 120-480 W for 30 min reduced Alicyclobacillus acidoterrestris spores by approximately 1 log, and that ultrasound treatment at 5000 W and 80% amplitude for 10 min inactivated nutrient cells of Anoxybacillus flavithermus and Bacillus coagulans in skim milk by 4.26 and 4.53 log, respectively. However, further studies are needed to determine the optimal treatment conditions and the corresponding mechanisms of inhibition [13,14]. Therefore, the aims of this study were to investigate the inhibitory effect of ultrasound on Bacillus subtilis and to examine the inhibitory mechanisms using label-free quantitative proteomics. ...
... These results were similar to those of previous studies. Researchers found that A. flavithermus decreased by 4.26 log after sonication for 10 min in optimal conditions and that B. coagulans decreased by 4.53 log; the log reduction of both Bacillus species increased significantly with longer sonication times [14]. In contrast, Yu et al. found that ultrasound at 60 W and 10 min treatment killed only<0.1 log of Staphylococcus aureus; this may have been due to the protective effect of the biofilm on bacteria, which mitigates the direct lethal effect of ultrasound [26]. ...
... The transient cavitation effect induced by HIU and the accompanying shock waves and liquid jets causes the perforation and rupture of the bacterial cell wall/membrane; this is generally considered to be the main mechanism of ultrasound-induced bacterial damage [29]. A previous study reported that ultrasound treatment had strong mechanical destructive effects on both nutrient cells and spores of some Bacillus species [14]. Similar effects were also found in this study (Fig. 2B-D). ...
Article
Full-text available
The bacteriostatic effects of high-intensity ultrasonic treatment (HIU) on Bacillus subtilis vegetative cells were evaluated, and the related mechanisms were explored using quantitative proteomics. The bacteriostatic effect of HIU on B. subtilis was proportional to the ultrasound treatment time and power, and the number of cultivable B. subtilis cells was decreased by approximately one log (at 270 W for 15 min) or half log (at 90 W for 25 min or 360 W for 5 min). Scanning electron microscopy images and gel electrophoresis results showed that HIU caused the destruction of the cell structure and intracellular protein leakage. In addition, HIU treatment at 270 W for 15 min resulted in the greatest decrease (84.22%) in intracellular adenosine triphosphate (ATP) content. The quantitative proteomic analysis showed that B. subtilis resisted the stress of HIU treatment by regulating the key proteins in physiological activities related to membrane transport (ATP-binding cassette [ABC] transporter), signal transduction (the two-component system), and energy metabolism (the tricarboxylic acid [TCA] cycle). HIU-induced physical damage, stress, and metabolic disorders were the main causes of the bacteriostatic effects on B. subtilis. These findings provide a foundation for the subsequent optimization and potential applications of HIU inactivation of B. subtilis.
... These techniques have shown the ability to kill vegetative microorganisms and some spores Piyasena et al., 2003). In fact, thermoduric aerobic sporeformers microbes are able to survive milk pasteurization and cause spoilages of dairy products (Gopal et al., 2015;Khanal et al., 2014a). Moreover, these thermal processes required high levels of energy, so they impact on the nutritional content, sensory properties, and final products quality Piyasena et al., 2003). ...
... Moreover, these thermal processes required high levels of energy, so they impact on the nutritional content, sensory properties, and final products quality Piyasena et al., 2003). However, alternative technologies of food processing and preservation, such as ultrasound have been explored in the last years (Higuera- Barraza et al., 2016;Khanal et al., 2014a;Yanjun et al., 2014). High-intensity, low-frequency, or power ultrasound (10-1000 W cm 2 or 20-100 kHz) generates acoustic cavitation in a liquid medium. ...
... Several studies have shown the ability of ultrasound to inactivate spoilage and pathogenic microorganisms and enzymes in dairy products (Gao et al., 2013(Gao et al., , 2014Mohammadi et al., 2014;Pingret et al., 2013;Şengül et al., 2011). According to Khanal et al. (2014a), traditional pasteurization was ineffective in inactivating the vegetative cells of microbes associated with spoilage in milk, such as Bacillus coagulans and Anoxybacillus. To protect the rights of the author(s) and publisher we inform you that this PDF is an uncorrected proof for internal business use only by the author(s), editor(s), reviewer(s), Elsevier and typesetter Thomson Digital. ...
... The use of ultrasound combined with other anti-microorganism technologies, such as heat (that is thermosonication, TS), ultraviolet light, and antibacterial agents, is often recommended owing to low inactivation rate and high-energy cost of ultrasound [10]. Most studies on microbial inactivation by TS have shown a synergistic sterilization reaction in various food matrices, such as ayran [2], carrot juice [11], milk [12], and powdered infant formula [13]. There was not only encouraging results in microbial inactivation but also in preservation the nutritional quality of fresh products. ...
... Carrot juice sonicated at 58°C had the best quality; it retained more than 98% of carotenoids and 100% of ascorbic acid, and microbial growth remained low at ∼3-log for mesophiles, 4.5-log for yeasts and molds (Y&M), and 2-log for enterobacteria after 20 days of storage [11]. The combined treatment of pasteurization (63°C for 30 min) following by ultrasonication reduced approximately 6-log CFU/mL of the vegetative cells Bacillus coagulans and Anoxybacillus flavithermus in skim milk and failed to affect its pH and alkaline phosphatase activity [12]. ...
Article
The effects of thermosonication (TS) and 100 ppm nisin-assisted TS (TS + nisin) on the inactivation of naturally occurring microorganisms, retention of nutritional quality and extension of shelf life of fresh apple juice were evaluated, with nisin and mild heat (nisin + MH) treatments as control. Fresh apple juice was addressed by nisin + MH, TS and TS + nisin at 37, 42, 47, and 52 °C for 5–40 min. After processing, microbial growth was evaluated during storage at 8 °C at every 5 days. Temperature played a vital role in the inactivation of aerobic bacteria and yeasts and molds by TS and TS + nisin, higher temperature up to 52 °C could cause a considerable inactivation of microbial cells in apple juice. As apple juice was subjected to TS and TS + nisin at 52 °C for 30 min, retention of original quality including 89% ascorbic acid, non-visible color change, no significant alteration in BD, pH, TA and TSS values of fresh apple juice, and extension shelf life to 15 d at 8 °C were obtained. Nisin exhibit additional inactivation effect of aerobic bacteria in apple juice while not obviously effect on yeast and molds. These results indicated a potential application of TS and TS + nisin (100 ppm) to produce fresh-like quality apple juice and/or to extend its shelf life.
... A. flavithermus has an optimum growth temperature at 60 and 65 °C under aerobic and anaerobic conditions, respectively. G. stearothermophilus resists and grows at temperatures up to 75 °C, and its spores have the potential to survive UHT treatments (Khanal et al., 2014). Both exhibit a fast growth rate and tend to form biofilm on the stainless steel surfaces of processing equipment (i.e. ...
... High-pressure treatments also have been assayed to inactivate B. subtilis and B. cereus spores in cheese (Capellas et al., 2000;López et al., 2003). A combination of ultrasonication and pasteurisation resulted in complete inactivation of the thermally resistant vegetative cells of B. coagulans and A. flavithermus in milk (Khanal et al., 2014). ...
... Yet in some applications, ultrasound has been used to stimulate bacterial growth. Khanal et al. (2014b) reported that increased ultrasonication amplitude might induce sporulation rather increase endospore inactivation level. Low, sublethal doses of ultrasound can bust up clumps of cells, increasing total counts or colony-forming units (Marchesini et al., 2015). ...
... The localized stress potentially induced conditions for sporulation and later germination. Khanal et al. (2014b) applied this theory to ultrasonication and found similar results, as the treatments simply led to sporulation rather than destroying cells. ...
Article
High-power, low-frequency ultrasound has been suggested as a novel processing technique with the potential to extend milk shelf life via inactivation of bacteria and spores that survive standard pasteurization. The primary objective of this research was to determine whether short-duration (≤60 s) sonication treatment, in conjunction with pasteurization, can increase shelf life while producing no adverse aroma effect. Skim milk was inoculated with Paenibacillus amylolyticus, a spore-forming, thermotolerant and psychrophilic milk contamination bacterium. Milk was sonicated under 6 selected amplitude and time conditions, except for control. Both cold sonicated (C-S) and thermosonicated (T-S) milk and milk treatments were pasteurized; however, T-S milk was sonicated after pasteurization (72.5 ± 0.3°C; mean ± SD), whereas C-S milk was sonicated at 12.5 ± 5°C (mean ± SD) before pasteurization. Milk was refrigerated up to 50 d and total aerobic counts were enumerated on pasteurized control, C-S, and T-S milk weekly. Neither C-S nor T-S treatments reduced total aerobic counts to an equivalent level as pasteurization alone. Counts in pasteurized controls and C-S milk did not exceed 3.00 log cfu/mL for up to 50 d; counts in T-S milk exceeded 5.00 cfu/mL by d 36. Aroma qualities (cooked, lacks freshness, and rubbery) of 2 T-S treatment intensities [170 µm peak-to-peak (p-p) for 60s and 200 µm p-p for 10 s] and pasteurized controls were evaluated by a trained descriptive sensory panel. No significant differences were observed in cooked or lacks freshness aromas among samples. Only the milk treated with 170 µm p-p for 60 s had significantly higher rubbery aroma on d 1 compared with milk treated with 200 µm p-p for 10 s. Although the sensory effects of T-S on milk may not limit the commercial feasibility of cold sonication or thermosonication, conditions that differ from those used in the present study should be considered in the future. Neither C-S nor T-S were appropriate techniques for reducing bacterial count in fluid milk beyond standard pasteurization and, in fact, increased counts of spore-forming spoilage bacteria.
... The effects of US on milk enzymes are presented in Table 1. Different researchers have studied the effects of different ultrasonic treatments on the inactivation of milk enzymes (Khanal et al. 2014). Generally, the enzyme activity decreased with the increase in the enzyme concentration, while higher solid content (i.e. ...
... The LPO and AP activities were not affected with the increase in the residence time to 102.3 sec, while a reduction of 17.2% was observed in the c-GTP activity (Villamiel and de Jong 2000). Khanal et al. (2014) reported similar results for the AP activity, with no significant reduction after 1 and 5 min of treatment time (amplitude 80%, 20 kHz), with 5.8% reduction after 10 min of process. The activity of bile salt-stimulated lipase (BSSL) in human milk was investigated at a constant US energy (1000 J) and US power of 3.62 W (Christen et al. 2012). ...
... A. flavithermus has an optimum growth temperature at 60 and 65 °C under aerobic and anaerobic conditions, respectively. G. stearothermophilus resists and grows at temperatures up to 75 °C, and its spores have the potential to survive UHT treatments (Khanal et al., 2014). Both exhibit a fast growth rate and tend to form biofilm on the stainless steel surfaces of processing equipment (i.e. ...
... High-pressure treatments also have been assayed to inactivate B. subtilis and B. cereus spores in cheese (Capellas et al., 2000;López et al., 2003). A combination of ultrasonication and pasteurisation resulted in complete inactivation of the thermally resistant vegetative cells of B. coagulans and A. flavithermus in milk (Khanal et al., 2014). ...
Chapter
Various spore forming bacteria belonging to Bacilli and Clostridia classes, which include pathogenic and spoilage associated species, are among the most resistant life forms known. Spores are dormant cellular structures in the life cycle of spore forming bacteria, much more resistant to environmental challenges than vegetative cells of the same species due to a variety of factors related to spore structure and composition. Spore forming bacteria of Bacillus spp. and related genera, and of Clostridium spp., are important contaminants in the dairy industry since they are ubiquitous in nature, can enter the milk chain from different sources (i.e. farm environment, raw milk, dairy plant equipment), can attach to processing equipment and form biofilms, are highly resistant to heat, desiccation or disinfectants, and significantly affect product safety and quality. Certain spore formers like Bacillus cereus, Clostridium botulinum and Clostridium perfringens pose a risk of causing dairy product poisoning by the production of toxins. Moreover, contamination with spore formers can lead to spoilage of milk and dairy products, mainly caused by enzyme deterioration (proteolytic and lipolytic activity by Bacillus species), acid production, (i.e. lactic, butyric and acetic acid), gas production (CO2+ H2 gas by Clostridium species) or exceeding number specifications. Diverse preventive and palliative control strategies to prevent poisoning and spoilage of milk and dairy products are adopted by farmers and at the dairy industry (i.e. good farming practices, physically removing of spores, addition of preservatives to avoid spore germination, use of physical, biological and chemical antimicrobial treatments) although the risk of contamination with spores is impossible to eliminate.
... It had been shown in an early study that ultrasound processing with varying times (up to 45 min) under different temperatures (40-85°C) produced more extensive changes of the secondary structure and surface activity of bovine serum albumin than ultrasound alone [18]. In fact, a similar thermosonication approach has been taken to inactivate microorganisms in beverage [19,20] thermophilic spore-forming bacteria and extend product shelf-life without compromising bioactive compounds and the nutritional quality of the product [21,22]. ...
... Furthermore, with the increase of heating temperature and time, the amount (band intensity) of extensively crosslinked polymers, shown at the top of both stacking and separating gels, Fig. 6. SDS-PAGE of the soluble fraction of mung bean protein isolate treated by heating alone or ultrasound combined with heating at different processing times (5,10,20, and 30 min) and controlled temperatures (30°C, 50°C, and 70°C). Samples with (+βME) or without (-βME) 5% β-mercaptoethanol are indicated. ...
Article
Mung bean protein is considered a highly nutritive food ingredient, but its solution properties are not well defined. In this study, suspensions of mung bean protein isolate (MPI, 10%, w/v) were subjected to high intensity ultrasound (20 kHz, 30% amplitude) at varied durations (5, 10, 20, and 30 min) with controlled temperatures (30, 50, and 70 °C) to determine the effects of thermosonication treatment on physical properties of the protein solution. Results showed that thermosonication treatment significantly reduced the particle size and free sulfhydryl content of MPI in a time-dependent manner. Ultrasound increased surface hydrophobicity, and the exposure of nonpolar groups led to the formation of soluble aggregates. Changes in secondary structure of MPI were minimal at 30 and 50 °C but were significant at 70 °C. The dissociation of native components followed by reaggregation into soluble particles following ultrasound treatment at 70 °C resulted in remarkable improvements of protein solubility (>2 fold), clarity, and stability of the MPI suspensions. The findings indicated that thermosonication could be a promising technology for the processing of mung bean protein beverage.
... Ansari, Ismail, and Farid (2017) reduced 2 log CFU g −1 of B. subtilis spores in whole milk using an ultrasound treatment (1.1 W mL −1 ) combined with heat treatment to 100°C. Khanal, Anand, Muthukumarappan, and Huegli (2014) reduced 4.5 log CFU mL −1 of B. coagulans in skim and whole milk by high-intensity ultrasound combined with thermal treatment. In summary, the literature of the spore formers inactivation by ultrasonic involves prolonged exposure time (>5 min), which limits the applicability of ultrasound at an industrial level. ...
... A suspension of vegetative cells of Bacillus coagulans (ATCC 12245) was prepared as described by Khanal et al. (2014). Briefly, B. coagulans was grown in Brain Heart Infusion (Difco, Becton, Dickinson and Company, Sparks, NV, USA) at 37°C for 8 h under aerobic conditions. ...
Article
Full-text available
We report the development of hydrodynamic cavitation coupled with thermal treatment for reducing counts of B. coagulans (ATCC 12245) in skim milk concentrate (SMC, 35 ± 1% total solids). The process was evaluated in terms of analysis of cavitation parameters, characterization of the increase in the fluid temperature, and microbial inactivation. Inoculated SMC (4.67 ± 0.18 log) was treated by cavitation (rotor speed of 3600 RPM and fluid flow rate of 100 L h⁻¹), thermal treatment (75–85 °C for 14–106 s), or a combined cavitation-thermal treatment, using the aforementioned conditions. A 3.5 log CFU mL⁻¹ reduction was obtained by the combined cavitation-thermal treatment, while thermal treatment alone yielded a 2.77 log CFU mL⁻¹ reduction. Contrary, individual cavitation did not produce a significant reduction. The values of decimal reduction time at 75 °C (D75) were lower when cavitation was followed by thermal treatment (375 ± 62 s) than the values obtained for thermal treatment (470 ± 66 s). The outcomes of this study present opportunities for reducing the counts of B. coagulans in skim milk concentrate by the combined effect of hydrodynamic cavitation and thermal treatment.
... Use of ultrasound as an alternative method of processing dairy products has been explored in recent years and examined in terms of physical and functional properties of the food product, food safety, shelf life and possible economic savings to the food producer [24][25][26][27]. Emulsification of milk products is of interest and several studies have examined the effect of ultrasound on the degree of emulsification and the overall homogenization of the final drink [28,29]. ...
... Several studies have shown the ability of ultrasound to inactivate spoilage and pathogenic microorganisms and enzymes in dairy products [6,24,25,[76][77][78][79] and fruit juices [80,17,81,82] (See Table 3 and below for further details). This is often attributed to thinning of cell wall membranes, formation of localized hotspots and production of free radicals within the bulk medium [83][84][85]. ...
Article
Ultrasonic processing of a variety of liquids, drinks and beverages has generated much interest with published literature papers increasing within this area in recent years. Benefits include enhanced emulsification with improved homogenization and fat globule size reduction being recorded. In dairy systems increased creaming rates are observed on sonication in a process known as fractionation. Whilst fruit juices exhibit retention or enhancement of quality parameters whilst increasing levels of bioactive compounds. Sterilization of liquids is a large feature of ultrasonic treatment with microbial activity of a range of fruit juices being monitored over time as increased stability and reduced spoilage is observed. Progress has also been made towards scale up of ultrasonic processes with several examples of batch and continuous processes being studied with reduced processing times and temperatures being quoted as a result of ultrasonic treatment. This short review covers the effect of sonication on liquids and beverages with a specific focus towards dairy and fruit juices and covers emulsification, fractionation, sterilization and some pilot scale initiatives.
... The resulting pellets were subsequently diluted in PBS at pH 7.4, and maintained in 1.8-mL cryogenic vials containing sterile beads and glycerol (Cryobank, Copan Diagnostic Inc., Murrieta, CA). The vials were stored in a deep freezer (NuAire Ultralow freezer, NuAire Inc., Plymouth, MN) at −80°C for future experiments (Khanal et al., 2014). Before use, the pellets were suspended in PBS, and final suspensions adjusted to a concentration of 1 × 10 7 cfu/mL. ...
... The contents in the tube were mixed and appropriate serial dilutions were made with sterile PBS at pH 7.4. Aerobic plate counts were performed on BHI agar plates (Khanal et al., 2014) using the spread plate technique (Downes and Ito, 2001). Colonies that appeared on the agar plates were counted after 24 h of incubation at the optimum growth temperature of the bacteria. ...
Article
The attachment of aerobic spore-forming bacteria and their spores to the surfaces of dairy processing equipment leads to biofilm formation. Although sporeformers may differ in the degree of attachment, various surface modifications are being studied in order to develop a surface that is least vulnerable to attachment. This study was conducted to compare the extent of adhesion of spores and vegetative cells of the thermotolerant sporeformer Bacillus licheniformis and the high-heat-resistant sporeformers Geobacillus stearothermophilus and Bacillus sporothermodurans on both native and modified stainless steel surfaces. We studied the effect of contact surface and cell surface properties (including surface energy, surface hydrophobicity, cell surface hydrophobicity, and zeta potential) on the adhesion tendency of both types of sporeformers and their spores. Attachment to native and modified (Ni-P-polytetrafluoroethylene, Ni-P-PTFE) stainless steel surfaces was determined by allowing interaction between the respective contact surface and vegetative cells or spores for 1 h at ambient temperature. The hydrophobicity of vegetative cells and spores of aerobic spore-forming bacteria was determined using the hexadecane assay, and zeta potential was determined using the Zeta sizer Nano series instrument (Malvern Panalytical, Malvern, UK). The results indicated a higher adhesion tendency of spores over vegetative cells for both thermotolerant and high-heat-resistant sporeformers. On comparing the sporeformers, B. sporothermodurans demonstrated the highest adhesion tendency followed by G. stearothermophilus; B. licheniformis exhibited minimal attachment on both surfaces. The tendency to adhere varied with cell surface properties, decreasing with lower cell surface hydrophobicity and higher cell surface charge. On the other hand, modifying contact surface properties for higher surface hydrophobicity and lower surface energy decreased attachment.
... The pellets thus obtained were subsequently suspended in PBS at pH 7.4 and maintained in 1.8-mL cryogenic vials (Copan Diagnostic Inc., Murrieta, CA) that contained sterile beads and glycerol. The vials were stored in a NuAire ultralow deep freezer (NuAire Inc., Plymouth, MN) at −80°C until further use (Khanal et al., 2014). ...
... The contents in the tube were then mixed and serially diluted in sterile PBS solution at pH 7.4. Aerobic plate counts were performed on BHI agar plates (Khanal et al., 2014) using spread plate technique (Downes and Ito, 2001). The plates were incubated for 24 to 48 h at 50°C. ...
Article
The development of bacterial biofilms on stainless steel (SS) surfaces poses a great threat to the quality of milk and other dairy products as the biofilm-embedded bacteria can survive thermal processing. Established biofilms offer cleaning challenges because they are resistant to most of the regular cleaning protocols. Sporeforming thermoduric organisms entrapped within biofilm matrix can also form heat-resistant spores, and may result in a long-term persistent contamination. The main objective of this study was to evaluate the efficacy of different nonfouling coatings [AMC 18 (Advanced Materials Components Express, Lemont, PA), Dursan (SilcoTek Corporation, Bellefonte, PA), Ni-P-polytetrafluoroethylene (PTFE, Avtec Finishing Systems, New Hope, MN), and Lectrofluor 641 (General Magnaplate Corporation, Linden, NJ)] on SS plate heat exchanger surfaces, to resist the formation of bacterial biofilms. It was hypothesized that modified SS surfaces would promote a lesser amount of deposit buildup and bacterial adhesion as compared with the native SS surface. Vegetative cells of aerobic sporeformers, Geobacillus stearothermophilus (ATCC 15952), Bacillus licheniformis (ATCC 6634), and Bacillus sporothermodurans (DSM 10599), were used to study biofilm development on the modified and native SS surfaces. The adherence of these organisms, though influenced by surface energy and hydrophobicity, exhibited no apparent relation with surface roughness. The Ni-P-PTFE coating exhibited the least bacterial attachment and milk solid deposition, and hence, was the most resistant to biofilm formation. Scanning electron microscopy, which was used to visualize the extent of biofilm formation on modified and native SS surfaces, also revealed lower bacterial attachment on the Ni-P-PTFE as compared with the native SS surface. This study thus provides evidence of reduced biofilm formation on the modified SS surfaces.
... These cavities keep absorbing acoustic energy until they reach a critical size and break violently, releasing high energy. This phenomenon is called the cavitation effect, which is favored by the food industries for its excellent cleaning, processing and disinfection performance (Khanal, Anand, Muthukumarappan, & Huegli, 2014). ...
... The color of food is an important factor of sensorial quality, which directly affects the purchase decision of consumers. Continuous ultrasound can cause non enzymatic browning of food, and brown matter increased with the processing time (Khanal et al., 2014). On the other hand, the free radicals produced by continuous ultrasound may degrade the pigments in fruit juice. ...
Article
Background The existence of chemical contaminants in food brings a serious threat to human health. Researchers have been making persistent efforts to eliminate contaminants from food to make it safe for human consumption. Traditional methods, such as washing with various agents, peeling, cooking and chemical oxidants, cannot achieve the desired results. In recent years, non-thermal technology, such as ultrasound, has attracted extensive attention as an emerging technology for removing food contaminants. Scope and approach This review analyzed the research status of ultrasound in the elimination of food contaminants, studied the transformation products and their potential toxicity during ultrasound treatment, discussed the effect of ultrasound on food quality, and summarized the advantages and disadvantages of ultrasound treatment. At the same time, the challenges of this technology, the problems to be studied, and future development prospects were discussed. Key findings and conclusions Ultrasound is a green processing technology, which would not impart secondary pollution. It also has advantages of high efficiency and low energy consumption, compared with traditional decontamination technologies. The combination of ultrasound with other techniques such as ultraviolet, ozone and pulsed electric field shows better decontamination effect. However, ultrasound treatment may cause degradation of some phenolic compounds and vitamins, changes in color, loss of anthocyanin and other adverse effects on food characteristics. In addition, the appropriate ultrasound processing system in terms of probe design, geometry, and operating conditions, needs to be specially designed for different food materials. Thus, some challenges need to be addressed to improve its application.
... Inactivation of bacteria is done via low frequency ultrasound (also called high intensity or power ultrasound) applied in a liquid medium, whereby power levels of 10-1000 W/cm 2 are generated resulting in cavitation (Raso et al., 1998;Villamiel et al., 2009). One of the latest studies on ultrasonication applied to thermoduric aerobic sporeformers has determined that high intensity power ultrasonication is able to inactivate microorganisms within 10 min and, when combined with pasteurization, all vegetative microbial cells are inactivated (Khanal et al., 2014b). The same research group has also been investigating the actions of ultrasonication on spores of B. coagulans, B.licheniformis, and G. stearothermophilus. ...
... The same research group has also been investigating the actions of ultrasonication on spores of B. coagulans, B.licheniformis, and G. stearothermophilus. Their studies determined that high intensity ultrasonication combined with pasteurization inactivated 65.74% of endospores present in non-fat milk while ultrasonication combined with a heat treatment at 80 • C for 1 min inactivated 75.32% of endospores (Khanal et al., 2014b). Despite being a powerful tool as it can reach biofilms in cracks and crevices, ultrasonication alone cannot completely dislodge all biofilms. ...
Article
Full-text available
Milk produced in udder cells is sterile but due to its high nutrient content, it can be a good growth substrate for contaminating bacteria. The quality of milk is monitored via somatic cell counts and total bacterial counts, with prescribed regulatory limits to ensure quality and safety. Bacterial contaminants can cause disease, or spoilage of milk and its secondary products. Aerobic spore-forming bacteria, such as those from the genera Sporosarcina, Paenisporosarcina, Brevibacillus, Paenibacillus, Geobacillus and Bacillus, are a particular concern in this regard as they are able to survive industrial pasteurization and form biofilms within pipes and stainless steel equipment. These single or multiple-species biofilms become a reservoir of spoilage microorganisms and a cycle of contamination can be initiated. Indeed, previous studies have highlighted that these microorganisms are highly prevalent in dead ends, corners, cracks, crevices, gaskets, valves and the joints of stainless steel equipment used in the dairy manufacturing plants. Hence, adequate monitoring and control measures are essential to prevent spoilage and ensure consumer safety. Common controlling approaches include specific cleaning-in-place processes, chemical and biological biocides and other novel methods. In this review, we highlight the problems caused by these microorganisms, and discuss issues relating to their prevalence, monitoring thereof and control with respect to the dairy industry.
... Pasteurization has been used for decades to kill pathogenic bacteria (Farkas, 1998). We have conducted studies to combine thermal and nonthermal techniques to inactivate or kill common spore formers by applying ultrasonication and pasteurization (Khanal et al., 2014). Although these treatments are quite effective at inactivating bacteria, unfortunately, some of the cells are injured but able to recover.. Exposing common spore formers to several nonthermal treatments such as ultrasonication affects the viability of sporeformers (Wu et al., 2000) and leads to injury and later recovery in dairy products (Deeth and Datta, 2011). ...
... However, B. licheniformis and B. sporothermodurans showed counts of 4.38 ± 0.021 and 3.75 ± 0.055 log cfu/mL under the same processing parameters. The extent of reduction in spore former counts as a result of ultrasonication was comparable to that of our previous study (Khanal et al., 2014). ...
Article
Full-text available
The presence of spore-forming microorganisms and their adhesion to contact surfaces in dairy plants is a major concern because dairy products are prone to cross-contamination. Spore formers and their spores can survive milk processing treatments due to their heat resistance. One source of these spore formers is bacterial biofilms, which grow and accumulate on most surfaces in dairy industrial plants, such as pipes, heat exchangers, pasteurized plates, and storage tanks. Their endospores form biofilms by attaching to these surfaces and germinating when conditions become more favorable. The cross-contamination of dairy products by bacterial biofilms may lead to reduced shelf life and spoilage. To minimize the problem caused by thermoduric bacteria, the dairy industry adopts several approaches. Pasteurization is an irreplaceable technique for milk processing. Unfortunately, some bacteria and endospores are resistant to heat treatment, which can grow and cause spoilage of dairy products. Thus, alternative approaches that could help to reduce microbial counts are needed. In our previous study, we demonstrated the effectiveness of ultrasonication to inactivate spore formers and reduce the overall microbial counts in milk. In the current study, we investigated the recovery of cavitation-induced injured cells during the storage of ultrasonicated skim milk. Three common spore formers—Geobacillus stearothermophilus (ATCC 15952), Bacillus licheniformis (ATCC 6634), and Bacillus sporothermodurans (DSM 10599)—were selected to conduct challenge studies by inoculating skim milk samples and exposing them to ultrasonication (10 min each at 80% amplitude). This treatment was done in an ice bath to control the resultant temperature increase. The ultrasonicated skim milk samples were then held for 1, 2, 4, or 12 h in the refrigerator (4°C) to study the recovery of cells following cavitation-induced injury. Ultrasonication resulted in cell injury, as demonstrated by scanning electron microscopy. The injured cells can potentially recover under appropriate conditions during the storage of ultrasonicated milk and could affect the microbiological quality of milk and products manufactured with such milk. The respective bacterial counts for the 3 organisms in the spiked skim milk, on average, were approximately 6.0 log cfu/mL; immediately after ultrasonication, these counts decreased to 3.50 ± 0.02, 4.38 ± 0.02, and 3.75 ± 0.05 log cfu/mL for G. stearothermophilus, B. licheniformis, and B. sporothermodurans, respectively. During 12 h of subsequent incubation at 4°C, their counts increased to 4.17 ± 0.05, 5.25 ± 0.1, and 5.69 ± 0.06 log cfu/mL, respectively. All experiments were done in triplicate for all 3 bacteria. To conclude, slow recovery of injured cells of spore-forming bacteria is possible in ultrasonicated milk during storage under refrigeration conditions.
... For 65 C a calculation of the D-values from the measured points varies for the first three points from 0.54 min to 1,88 min of the last three points. For the vegetative cells of A. flavithermus Khanal, Anand, Muthukumarappan, and Huegli (2014) showed that a temperature treatment at 60 C for 30 min (long-term pasteurization) achieved a negligible reduction of 0.03 log-levels and thus the tested strain can survive the pasteurization (Kessler, 2002)) in its vegetative form, but would sur- vive a pasteurization at 60 C for 30 min as well. The z-value for living A. flavithermus F48 cells was calculated to be 3.4 K. ...
... Sanz et al. (1985) also observed a decrease in the heat resistance of Geobacillus stearothermophilus spores after ultrasonic treatment at room temperature. Additionally, Bacillus coagulans inactivation by 6 log was observed in skimmed milk after pasteurisation (60 min, 30 min) followed by a 10 min ultrasonic pretreatment (20 kHz, 0°C, 250 W/mL) (Khanal et al., 2014). To date, the efficacy of ultrasound assisted thermal inactivation of Clostridium spores is under investigation. ...
... Goodenough and Solberg (1972) found no heat sensitizing effect on C. perfringens spores at 90 C after sonicating for 12 min (0 C, 15 W/mL). Another study showed a significant decrease in heat resistance of G. stearothermophilus spores after ultrasonic treatment at room temperature (Khanal, Anand, Muthukumarappan, & Huegli, 2014). Moreover, B. coagulans inactivation of 6 log was obtained using heat treatment (60 and 30 min) combined with a 10 min ultrasonic pretreatment (20 kHz, 0 C, 250 W/mL). ...
... UV-C treatment did not change the UV-C pretreatment (D Act 2.37 ± 0.126 J/ml) followed by a thermal treatment at 110 • C for 30 s resulted in a 1.1 log CFU/ml in sheep milk. Micromorphology (SEM) of Anoxybacillus flavithermus before and after ultrasonication treatment fromKhanal et al. (2014) plasma-clotting capability of DHM, US was shown to be almost equally detrimental as HTST, indicating that this technique is not completely harmless to human milk's bioactivity(Hu et al., 2022). ...
Article
Human milk and commercial dairy products play a vital role in humans, as they can provide almost all essential nutrients and immune‐active components for the development of children. However, how to retain more native immune‐active components of milk during processing remains a big question for the dairy industry. Nonthermal technologies for milk processing are gaining increasing interest in both academic and industrial fields, as it is known that thermal processing may negatively affect the quality of milk products. Thermosensitive components, such as lactoferrin, immunoglobulins (Igs), growth factors, and hormones, are highly important for the healthy development of newborns. In addition to product quality, thermal processing also causes environmental problems, such as high energy consumption and greenhouse gas (GHG) emissions. This review summarizes the recent advances of UV‐C, ultrasonication (US), high‐pressure processing (HPP), and other emerging technologies for milk processing from the perspective of immune‐active components retention and microbial safety, focusing on human, bovine, goat, camel, sheep, and donkey milk. Also, the detailed application, including the instrumental design, technical parameters, and obtained results, are discussed. Finally, future prospects and current limitations of nonthermal techniques as applied in milk processing are discussed. This review thereby describes the current state‐of‐the‐art in nonthermal milk processing techniques and will inspire the development of such techniques for in‐practice applications in milk processing.
... Generally, ultrasound treatment can modify rheological properties like reduction in bulk viscosity and increase in hydrophobicity of dairy proteins, e.g., whey protein concentrate, milk protein from retentate, and calcium caseinate, and can reduce the protein aggregate size. This should improve food safety, increase shelf life, and enable economic savings [81][82][83][84]. Hence, this method is considered to be industrially useful, but further work is still required since this technique has only been tested under laboratory conditions [85][86][87][88]. ...
... Only 1 milk sample (5) did not present any Bacillus or Paenibacillus isolates, meaning that 19 (95%) samples did present these genera. As an alternative for the control of these thermoduric microorganisms, the study by Khanal et al. (2014) reports that the ultrasonication of milk can eliminate the vegetative forms of the thermoduric microorganisms of the genus Bacillus. ...
Article
When correctly pasteurized, packaged, and stored, milk with low total bacterial counts (TBC) has a longer shelf life. Therefore, microorganisms that resist heat treatments are especially important in the deterioration of pasteurized milk and in its shelf life. The aim of this work was to quantify the thermoduric microorganisms after the pasteurization of refrigerated raw milk samples with low TBC and to identify the diversity of these isolates with proteolytic or lipolytic potential by RFLP analysis. Twenty samples of raw milk were collected in bulk milk tanks shortly after milking in different Brazilian dairy farms and pasteurized. The mean thermoduric count was 3.2 (±4.7) × 102 cfu/mL (2.1% of the TBC). Of the 310 colonies obtained, 44.2% showed milk spoilage potential, 32.6% were proteolytic and lipolytic simultaneously, 31% were exclusively proteolytic, and 48 (36.4%) were only lipolytic. Regarding the diversity, 8 genera were observed (Bacillus, Brachybacterium, Enterococcus, Streptococcus, Micrococcus, Kocuria, Paenibacillus, and Macrococcus); there was a predominance of endospore-forming bacteria (50%), and Bacillus licheniformis was the most common (34.1%) species. Considering the RFLP types, it was observed that the possible clonal populations make up the microbiota of different milk samples, but the same milk samples contain microorganisms of a single species with different RFLP types. Thus, even in milk with a high microbiological quality, it is necessary to control the potential milk-deteriorating thermoduric microorganisms to avoid the risk of compromising the shelf life and technological potential of pasteurized milk.
... Cameron et al. [27] reported on the efficiency of ultrasound treatment (20 kHz, 750 W and 4°C) on complete inactivation of various bacterial species such as E. coli after 10 min of sonication, Pseudomonas fluorescens in 6 min, and a 2-log reduction of Listeria monocytogenes in 10 min. Khanal et al. [28] described the effect of low-frequency ultrasound on the vegetative cells of spore forming bacteria such as Bacillus coagulans, Bacillus sporothermodurans, Bacillus licheniformis, A. flavithermus and G. stearothermophilus in milk. When only pasteurization treatment (63°C for 30 min) was applied to these thermoduric bacteria, B.licheniformis, B.coagulans and G. stearothermophilus were found to be susceptible to this treatment, whereas B.coagulans and A.flavithermus showed high resistance under the same condition. ...
Article
The vegetative cells and spores of Geobacillus spp. and Anoxybacillus flavithermus were subjected to 20 kHz ultrasound with a power ∼8 W. Ultrasonication had considerable effect on vegetative cells (5-log reduction in Geobacillus spp. and 1.6-log reduction in A.flavithermus). TEM imaging of the ultrasonicated vegetative cells showed an extensive damage both internally and externally. However, spores showed high resistance towards ultrasound treatment in the absence of NaOH and H2O2, although the outer layers such as the exosporium and the outer coat layer were disrupted, resulting in the reduced resistance of spores towards sonication. The combination of 0.12 M NaOH and 10 min ultrasonication inactivated 6 log spores of Geobacillus spp. A 7 log spores reduction of A.flavithermus was achieved by combining 0.17 M NaOH with 10 min ultrasonication. Ultrasonication combined with 1% H2O2 inactivated ∼7 log Geobacillus spp. spores in 6 min and ∼7 log A.flavithermus spores in 3 min. These ultrasound treatment in the presence of NaOH and H2O2 are synergistic as they showed a higher spores reduction when compared to NaOH combined with high temperature (85 °C), where only 1 and 3 log reduction was achieved in Geobacillus spp. and A.flavithermus spores, respectively.
... Levels of greater than 1000 cfu/ml are normally the result of poor cow hygiene and milking equipment (particularly in the case of ineffective hot wash routines). Potential sources of thermoduric bacteria include silage, faces, animal bedding, and soil [84]. Thermophilic bacteria grow in milk held at raised temperatures (55°C or higher), including pasteurization, 62.8°C, they include the Bacillus spp. ...
Chapter
Full-text available
Sampling and analysis occur along the milk processing train: from collection at farm level, to intake at the diary plant, the processing steps, and the end products. Milk has a short shelf life; however, products such as milk powders have allowed a global industry to be developed. Quality control tests are vital to support activities for hygiene and food standards to meet regulatory and customer demands. Multiples of chemical and microbiological contamination tests are undertaken. Hazard analysis testing strategies are necessary, but some tests may be redundant; it is therefore vital to identify product optimization quality control strategies. The time taken to undergo testing and turnaround time are rarely measured. The dairy industry is a traditional industry with a low margin commodity. Industry 4.0 vision for dairy manufacturing is to introduce the aspects of operational excellence and implementation of information and communications technologies. The dairy industries’ reply to Industry 4.0 is represented predominantly by proactive maintenance and optimization of production and logistical chains, such as robotic milking machines and processing and packaging line automation reinforced by sensors for rapid chemical and microbial analysis with improved and real-time data management. This chapter reviews the processing trains with suggestions for improved optimization.
... Levels of greater than 1000 cfu/ml are normally the result of poor cow hygiene and milking equipment (particularly in the case of ineffective hot wash routines). Potential sources of thermoduric bacteria include silage, faces, animal bedding, and soil [84]. Thermophilic bacteria grow in milk held at raised temperatures (55°C or higher), including pasteurization, 62.8°C, they include the Bacillus spp. ...
... In milk products, ultrasonication alone as a non-thermal processing technology is not explored, except few reports without heat application [8,9] and use of low temperatures [10,11]. On the other hand, processing under elevated ultrasonic powers and temperatures has been found to induce physical-chemical degradation and sensory unacceptability of milk and milk products [12,13]. ...
Article
Ultra-sonication (US) at varying intensities (200 W, 300 W and 400 W) and hydrodynamic cavitation (HC) at increasing pressures (6 bar, 8 bar and 10 bar) on freshly extracted peanut milk as non-thermal processing of milk for enhanced quality. The effects of US and HC was investigated on physico-chemical properties of peanut milk, microbial inactivation (total plate count and yeasts and molds), microstructure by optical microscopy and particle size, ζ-potential, sedimentation index, rheology and color measurements. The high temperature short time (HTST) treated milk samples have shown 1.53 and 2 log reduction in TPC, yeast and molds respectively with highest protein hydrolysis of 15.7%. Among the non-thermal treatments HC has shown highest log reduction of TPC at around 1.2 for sample treated at 10 bar pressure, whereas the US treatment was most effective for yeast and mold at 400 W with log reduction of 0.9. A non-Newtonian flow behaviour was observed for all peanut milk samples. Viscosity determined by Herschel-Bulkley equation decreased significantly (p > 0.05) after both cavitation treatments. The US was found to be superior to HC and HTST with improved separation index and colour attributes. Therefore, the US and HC appear to be a remarkable non-thermal processing methods for peanut milk and or any dairy or non-dairy beverages.
... Generally, ultrasound treatment can modify rheological properties like reduction in bulk viscosity and increase in hydrophobicity of dairy proteins, e.g., whey protein concentrate, milk protein from retentate, and calcium caseinate, and can reduce the protein aggregate size. This should improve food safety, increase shelf life, and enable economic savings [81][82][83][84]. Hence, this method is considered to be industrially useful, but further work is still required since this technique has only been tested under laboratory conditions [85][86][87][88]. ...
Article
The aim of this work was to prepare water‐in‐oil‐in‐water (w/o/w) emulsions using commonly available raw materials and a two‐step emulsification process. Several combinations of milk proteins and hydrocolloids were tested as internal water phases. Emulsions prepared with milk or colostrum exhibited the highest encapsulation efficiencies. In particular, w/o/w emulsions with internal water phases composed of colostrum, without addition of hydrocolloids, were the most stable. However, the addition of xanthan gum proved to be synergistic in stabilising w/o/w whey emulsions.
... For Non-Commercial Use same temperature [131]. The application of a sonication process (5000 W, 20 kHz) for 10 min following by pasteurization (63.5°C and 30 min) reached a complete inactivation of A. flavithermus and B. coagulans; while heat treatment and ultrasound applied alone of showed a lower microbial reduction [132]. In ayran (fermented milk) samples inoculated with S. thermophilus and L. delbueckii, a similar reduction (~ 7 log cycles) was observed between the thermosonication process (80°C for 1, 3 and 5 min) compared to thermal treatment (90°C for 1 min) for S. thermophilus, while L. delbrueckii was completely inactivated using thermosonication (80C for 5 min) and thermal (90°C for 1 min) treatment [133]. ...
... In the B. subtilis group, only one strain BS125 was positive for lecithinase activity, while the rest of the strains exhibited weak positive or negative results. Although lecithinase activity for aerobic sporeforming bacteria was mainly associated with strains from the B. cereus group, the strains from the B. subtilis group and P. polymyxa were sporadically reported to produce lecithinase (Fysun et al., 2019;Khanal et al., 2014;Oladipo et al., 2008). Lecithinase contributes to 'bitty cream' defects in milk by resulting in the aggregation of fat globules (Fysun et al., 2019). ...
... Color is an important factor in milk quality, which is related to the reflection of light by fat globules and casein micelles (Khanal et al., 2014). US/UV treatment of milk (Table S4) mainly influenced the CIE-LAB L* value (black/white) and b* value (blue/yellow); L* increased from 88.12 ± 0.12 to 90.22 ± 0.12 after 30 min. ...
Article
The aim of this study was to compare the degradation kinetics of chlorpyrifos by treatment with ultrasound (US), ultraviolet radiation (UV) and a combination of both (US/UV), to evaluate the toxicity of the degradation products and the effect of the treatments on milk quality. US/UV markedly accelerated the degradation of chlorpyrifos. The half-life of chlorpyrifos by US/UV was 6.4 min, which was greatly shortened compared to the treatment with US or UV alone. Five degradation products were identified by GC-MS, and a degradation pathway for chlorpyrifos was proposed, based on density functional theory calculations. According to the luminescent bacteria test and predictions from a structure/activity relationship model, the toxicity of the degradation products was lower than that of chlorpyrifos. In addition, US/UV treatment had little effect on the quality of the treated milk. Therefore, US/UV can be used as a potential non-thermal processing method to degrade pesticide residues in milk.
Article
Fluid foods are typically heat-treated to eliminate pathogens and reduce microbial counts. Coupling thermosonication with heat may reduce the microbial load in fluid foods and enhance the product quality during storage. This study evaluated the effect of thermosonication along with heat in a laboratory-scale continuous system, on the survival of Geobacillus stearothermophilus in milk, at two different settings (setting 1: 27.7 s total heating time with or without 11.9 s of sonication; setting 2: 20.3 s total heating time with or without 7.1 s of sonication). This study also investigated the effect of thermosonication along with heat on indigenous microbiota in raw milk; and milk quality was assessed by pH, free fatty acid (FFA) content, and casein/total protein (CN/TP) content during storage at the two different settings. Overall, thermosonication with heat resulted in higher log reductions for G. stearothermophilus; but, the reduction was not significant overall. Thermosonication with heat significantly decreased the indigenous microbiota in milk as compared to heat alone at both settings. Longer residence times (setting 1) had significantly higher log reductions at week 0, and treatment samples had significantly higher reductions than control during storage time at both the settings. Treatment samples at setting 1 had significantly higher pH and CN/TP, and lower FFA content at week 4, as compared to the control. Thermosonication using practical residence times along with heat may reduce milk microbiota. Results from this study need to be verified in a scale-up study employing pasteurization conditions.
Article
This study hypothesised that there may be induction of pitting corrosion or microbially-induced corrosion on stainless steel (SS) dairy-processing surfaces by biofilms of common milk sporeformers such as Bacillus sporothermodurans and Geobacillus stearothermophilus. Scanning electron microscopy (SEM) and micrographs generated from energy-dispersive spectroscopy were used to observe pitting corrosion and to find the elemental composition and distribution on the control and pitted surfaces. From SEM images and energy-dispersive spectroscopy micrographs, it was observed that pitting corrosion on SS could be induced by biofilms of both B. sporothermodurans and G. stearothermophilus.
Article
Increasing pasteurized milk shelf life has been a concern for the dairy industry. This study evaluated the effect of pasteurization (control) coupled with thermosonication (treatment) (11.1 s) in a continuous system on shelf‐life parameters during storage along with consumer liking. Treatment samples had significantly lower microbial counts than control during storage. Lower free fatty acid and higher pH and casein/total protein values were observed for treatment samples during storage. In a consumer panel, panelists commented on treatment samples having an unknown off‐flavor. This unknown flavor declined after 4 days and the consumer liking of treatment samples after 4 days in a second consumer panel was not significantly different than control. Thermosonication in a continuous system coupled with pasteurization successfully improved the overall quality of whole milk as compared to heat only. Future studies could investigate thermosonication conditions to minimize changes in milk sensory attributes. Thermosonication when applied with pasteurization improves the microbial quality of milk during storage and may extend the shelf life by 2 weeks. Increased shelf life could possibly lead to reduced milk loss on the consumer level by extending the time milk can be consumed. Scale‐up systems will need to evaluate the effect of thermosonication at specific residence times and sonication power levels on milk quality during storage and consumer acceptance.
Article
The objective of this work was to determine the effect of milk bactofugation on the counts and microbial diversity of mesophilic (MT), psychrotrophic (PT), and thermophilic (TT) thermoduric bacteria and its potential as a technological method to remove spoilage microorganisms resistant to pasteurization. Different batches of raw milk from 69 dairy farms divided into sets in 3 bulk tanks (A, B, C) were evaluated at different times during the technological process. As the raw milk was preheated (∼55°C) immediately before bactofugation (10,000 × g), the effect of bactofugation was estimated by comparing the counts in raw, preheated, and bactofuged milk. This centrifugation was sufficient to reduce the isolation of 88% of the MT in preheated milk. For PT, it was possible to verify a reduction of 72.5% in batch C. The TT were not recovered at higher detection limits (<5 cfu/mL). For diversity, 310 isolates were identified using a molecular approach; 15 species of contaminating thermoduric bacteria were identified from raw and preheated milk, and only 6 species were recovered in bactofuged milk. Only MT were recovered from the bactofuged milk, mainly the species Lysinibacillus fusiformis (61.7%) and Bacillus licheniformis (12.3%). Both species are known to be endospore-forming psychrotrophs and have proteolytic or lipolytic activity. The bactofugation of raw milk reduced the number of isolates of B. licheniformis, Bacillus toyonensis, Micrococcus aloeverae, and Aestuariimicrobium kwangyangense by 33, 43, 86, and 92%, respectively, and reduced the isolates of Macrococcus caseolyticus, Lysinibacillus varians, Carnobacterium divergens, Microbacterium hominis, Kocuria indica, Micrococcus yunnanensis, Gordonia paraffinivorans, Bacillus invictae, and Kocuria kristinae to undetectable levels. The results of this study indicate that bactofugation can be applied by the dairy industry to reduce pasteurization-resistant microorganisms in combination with prophylactic measures to prevent the contamination of raw milk by spores and vegetative forms of bacteria.
Article
Full-text available
To improve the microbial stability of mildly processed acidified sauces, which are threatened by acid-tolerant spore-forming spoilage bacteria (ATSSB), this study investigated the individual effect of pH, water activity (aw), temperature and organic acids on the growth from ATSSB spores and their spoilage potential. The pH of nine commercial sauces was gradually increased (3.8–5.6), and 81 strains were isolated from each sauce with the lowest pH allowing microbial growth. After heat treatment and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, ten selected strains were identified to the Bacillus subtilis and Bacillus cereus species group by 16S rRNA gene sequencing. The selected isolates with three acid-tolerant reference strains all exhibited enzymatic spoilage activity (amylase, protease, lipase or lecithinase) and had D95˚C -values between 0.8-6.4 min. None of the 13 strains grew below 10 °C, while the minimum growth pH varied from 4.1-5.4 and the minimum aw varied from 0.89-0.96. Moreover, the minimum inhibitory concentrations of benzoic, sorbic, acetic and lactic acids were 0.04–0.07, 0.05–0.10, 0.11–0.30 and 0.20–1.05 g/100g, respectively. This work demonstrated that the ATSSB spores were sensitive to organic acids and refrigeration, contributing to more accurate strategies to preserve the mildly pasteurized sauces with reduced acids and salts.
Chapter
As one of the rapidly developing emerging nonthermal techniques, ultrasound and/or combination with other strategies have been applied to enhance food quality and ensure food safety, especially in food with heat-sensitive characteristics. Ultrasound has received more and more attention of researchers and food manufactures in recent years. However, the responses of microorganisms to ultrasound treatments arise challenges to the development of this technology in food industry. Food-borne pathogens may be unaffected, be sublethally injured, survive in a viable but non-culturable state, or be inactivated when exposed to ultrasound treatments. The effects of ultrasound in the morphology, structure, and function of membranes, intracellular structure, enzymes, proteins, and DNA might be varied and relevant with the microbial state. Till now, limited study has explored the regulator mechanisms underlying food-borne pathogens response to ultrasound treatment, though several important genes have been found to contribute to the microbial ultrasound resistance. This chapter mainly focused on the cutting-edge advances and challenges in the application of ultrasound for controlling food-borne pathogens in food, with the consideration of studies in the last 10 years.
Article
Endospores of Bacillus licheniformis [American Type Culture Collection (ATCC) 6634], Bacillus coagulans (ATCC 12245), and Geobacillus stearothermophilus (ATCC 15952) were spiked in sterile nonfat milk, and subjected to high intensity batch ultrasonication treatment at different amplitudes (80 or 100%) and durations (1 to 10 min). Increasing the amplitude from 80 to 100% did not result in enhanced inactivation of G. stearothermophilus endospores. However, an increase in the duration of ultrasonication from 1 to 10 min significantly increased the inactivation of endospores of all 3 species. About 48.96% of the G. stearothermophilus endospores were inactivated by ultrasonication alone, whereas ultrasonication and pasteurization combined increased the inactivation to 65.74%. Inactivation of endospores could be further enhanced to 75.32% by ultrasonication and higher heat (80°C/1 min) combination. Endospores of B. licheniformis and B. coagulans were inactivated to a lesser extent compared with G. stearothermophilus spores. Ultrasonicated B. licheniformis endospores germinated in higher numbers when compared with untreated endospores resulting in their greater inactivation during the combined treatment. During microstructure imaging of ultrasonicated endospores, although no structural damage was noticed, they showed irregular shrinkage and wrinkles with surface coarseness. This may also have contributed to their reduced thermal resistance, in addition to sporulation.
Article
Full-text available
The effect of ultrasound treatment on milk homogenisation was studied. A microscope, equipped with a camera and ocular micrometer, was used to determine the diameters of fat globules and to evaluate the homogenisation efficiency of milk samples homogenised by using ultrasonic and conventional homogeniser. It was found that ultrasound treatment with high amplitude (power) had an important effect on milk homogenisation compared with conventional homogenisation. As exposure times and power levels increased, homogenisation efficiency also rose. The highest homogenisation efficiency and the smallest fat globule diameter were 3.22 and 0.725 μm at power level 100 (450 W) for 10 min, respectively. The fat globule diameters at a power level of 40 (180 W) for 10 min were similar to those of conventional homogenisation.
Article
Full-text available
Ultrasound techniques find use in the food industry in both the analysis and modification of foods. Microbial and enzyme inactivation are other applications of ultrasound in food processing. The use of ultrasound on its own in the food industry for bacterial destruction is currently unfeasible; however, the combination of ultrasound and pressure and/or heat shows considerable promise. The future of ultrasound in the food industry for bactericidal purposes lie in thermosonication, manosonication, and manothermosonication, as they are more energy-efficient and result in the reduction of microbial and enzyme activity when compared to conventional heat treatment. The use of ultrasound and combined technologies, mechanisms, and effects of ultrasound combinations are discussed in this review.
Article
Full-text available
This paper describes trends and technological advances in the dairy industry for extended shelf life milk. Demands for longer shelf life and wider distribution of milk and milk products have resulted in the development of processes and packaging concepts to increase the shelf life of these products in cold chain distribution. Factors important for the shelf life of milk are described in detail, and changes necessary from normal pasteurized production are suggested. Various processing methods such as microfiltration, injection and infusion heat treatments are described, and advantages and disadvantages of the different methods explained. In addition, the contamination sources from process to package are discussed, and the prevention of such recontamination in the filling machine is proposed. The paper describes existing and novel methods for sterilization of packaging material in the filling machine and points to possible future developments in this important market segment in the dairy industry.
Article
Full-text available
Survival of Bacillus cereus spores of dairy silo tank origin was investigated under conditions simulating those in operational dairy silos. Twenty-three strains were selected to represent all B. cereus isolates (n = 457) with genotypes (RAPD-PCR) that frequently colonised the silo tanks of at least two of the sampled eight dairies. The spores were studied for survival when immersed in liquids used for cleaning-in-place (1.0% sodium hydroxide at pH 13.1, 75 degrees C; 0.9% nitric acid at pH 0.8, 65 degrees C), for adhesion onto nonliving surfaces at 4 degrees C and for germination and biofilm formation in milk. Four groups with different strategies for survival were identified. First, high survival (log 15 min kill < or =1.5) in the hot-alkaline wash liquid. Second, efficient adherence of the spores to stainless steel from cold water. Third, a cereulide producing group with spores characterised by slow germination in rich medium and well preserved viability when exposed to heating at 90 degrees C. Fourth, spores capable of germinating at 8 degrees C and possessing the cspA gene. There were indications that spores highly resistant to hot 1% sodium hydroxide may be effectively inactivated by hot 0.9% nitric acid. Eight out of the 14 dairy silo tank isolates possessing hot-alkali resistant spores were capable of germinating and forming biofilm in whole milk, not previously reported for B. cereus.
Article
Full-text available
beta-Carotene biochemistry is a fundamental process in mammalian biology. Aberrations either through malnutrition or potentially through genetic variation may lead to vitamin A deficiency, which is a substantial public health burden. In addition, understanding the genetic regulation of this process may enable bovine improvement. While many bovine QTL have been reported, few of the causative genes and mutations have been identified. We discovered a QTL for milk beta-carotene and subsequently identified a premature stop codon in bovine beta-carotene oxygenase 2 (BCO2), which also affects serum beta-carotene content. The BCO2 enzyme is thereby identified as a key regulator of beta-carotene metabolism.
Article
Full-text available
The three-dimensional immages of free and intrasporangial spores produced by scanning electron microscopy show surface structures not visible by phase-contrast microscopy. Although fine surface detail is not elucidated by scanning electron microscopy, this technique does afford a definitive picture of the general shape of spores. Spores of Bacillus popilliae, B. lentimorbus, B. thuringiensis, B. alvei, B. cereus, and Sarcina ureae have varying patterns of surface ridge formation, whereas spores of B. larvae, B. subtilis, and B. licheniformis have relatively smooth surfaces.
Article
Full-text available
Bacteria that differentiate into highly heat-resistant endospores (HHRS strains) may survive ultrahigh-temperature treatment of milk and germinate in the final product. They do not noticeably spoil the milk and are nonpathogenic. The complete (>96%) 16S rRNA genes from three HHRS strains were identical, and phylogenetic analysis placed them alongside Bacillus firmus in the B. megaterium group of the genus Bacillus. Moreover, the approximately 550 nucleotides between regions U2 and U5 were invariant for seven HHRS strains. However, three cloned 16S rRNA genes from one HHRS strain, M215, showed marked size and sequence variations within the V1 and V2 regions. DNA reassociation assays confirmed the distinction between a reference HHRS strain and closely related members of the B. megaterium group, notably, B. firmus (30%), B. benzoevorans (28%), and B. circulans (20%). Ribotyping and pyrolysis mass spectrometry both indicated that the HHRS strains belong to a homogeneous, species-ranked taxon, an exception being strain TP1248, which is slightly atypical. The HHRS strains are unusual in that they grow poorly, if at all, on nutrient agar; good growth is obtained on brain heart infusion agar. On subculture, most HHRS strains form long, filamentous rods which stain unevenly in the Gram reaction. They are strictly aerobic and do not produce acid from sugars. We propose the name Bacillus sporothermodurans for these bacteria, which are phenotypically and phylogenetically distinct from other Bacillus species. The type strain is M215 (= DSMZ 10599).
Article
Full-text available
A new strictly anaerobic, alkaliphilic, moderately thermophilic, fermentative, spore-forming bacterium, strain K1T, was isolated from manure samples (pH 6-8). Cells were Gram-positive, straight, non-motile rods that grew at temperatures of 37-66 degrees C (optimum at 62 degrees C) and in a pH range of 8.0-10.5 (optimum at 9.5-9.7). The bacterium fermented D-glucose, sucrose, D-fructose, D-trehalose and starch as carbon and energy sources. It required vitamins and its growth is stimulated by yeast extract. The major metabolic products were H2 and acetate. Cells were catalase-negative and could reduce nitrate to nitrite. The G+C content of the DNA was 42.2 mol%. Based on the phenotypic properties and 16S rDNA sequencing and DNA-DNA hybridization data, strain K1T (= DSM 12423T = ATCC 700785T = VKM B-2193T) was assigned to the new genus Anoxybacillus gen. nov., as a representative of a new species, Anoxybacillus pushchinensis sp. nov. 'Bacillus flavothermus' strain d.y., which was found to be closely related to strain K1T, is described as Anoxybacillus flavithermus comb. nov. (type strain = d.y.T = DSM 2641T).
Article
Full-text available
Some species of bacteria produce colonies and spores which agglomerate in spherical clusters (Bacillus subtilis) and this serves as a protection for the organisms inside against biocidal attack. Flocs of fine particles e.g. clay can entrap bacteria which can also protect them against the biocides. It is because of problems such as these that alternative methods of disinfecting water are under active investigation. One such method is the use of power ultrasound, either alone or in combination with other methods. Ultrasound is able to inactivate bacteria and deagglomerate bacterial clusters or flocs through a number of physical, mechanical and chemical effects arising from acoustic cavitation. The aim of this study was to investigate the effect of power ultrasound at different powers and frequencies on Bacillus subtilis. Viable plate count techniques were used as a measure of microbial activity. Results showed a significant increase in percent kill for Bacillus species with increasing duration of exposure and intensity of ultrasound in the low-kilohertz range (20 and 38 kHz). Results obtained at two higher frequencies (512 and 850 kHz) indicated a significant increase in bacteria count suggesting declumping. In assessing the bacterial kill with time under different sonication regimes three types of behaviour were characterized: High power ultrasound (lower frequencies) in low volumes of bacterial suspension results in a continuous reduction in bacterial cell numbers i.e. the kill rate predominates. High power ultrasound (lower frequencies) in larger volumes results in an initial rise in cell numbers suggesting declumping of the bacteria but this initial rise then falls as the declumping finishes and the kill rate becomes more important. Low intensity ultrasound (higher frequencies) gives an initial rise in cell numbers as a result of declumping. The kill rate is low and so there is no significant subsequent decrease in bacterial cell numbers.
Article
Psychrotrophic bacteria have been recognized as a recurring problem in the refrigerated storage and distribution of fluid milk and cream and other perishable dairy products for several decades. Much emphasis has been focused on postpasteurization contaminants that are psychrotrophic, (e.g., Pseudornonas, Flavobacteriwn, and Alcaligenes spp.). Common sources of these gram-negative, non-sporeforming organisms are equipment surfaces and water supplies. Although these organisms are generally heat sensitive, many of their associated proteinases and lipases can withstand moderate to severe heat treatments and cause product deterioration. With the advance of improved control of postpasteurization contamination by nonheat-resistant psychrotrophs, more recent attention has been directed at psychrotrophic sporeformers and their potential impact on milk quality and shelf life properties. Heat-resistant psychrotrophs include members from the genera Clostridium, Arthrobacter, Microbacterium, Streptococcus, Corynebacterium, and Bacillus. However, the predominant microorganisms which comprise this category are Bacillus species. These bacteria can be introduced into milk supplies from water, udder and teat surfaces, or from soil and milkstone deposits on farm bulk tanks, pumps, pipelines, gaskets, and processing equipment. There is speculation that they can also be postpasteurization contaminants. When in the spore state, these microorganisms easily survive the typical range of pasteurization conditions with subsequent germination and outgrowth of vegetative cells. These organisms produce degradative enzymes (e.g., proteinases, lipases, and phospholipases) similar to those of non-sporeforming psychrotrophs. Enzymatic activity results in the development of objectionable flavor and quality defects in dairy products. The unique combination of both heat-resistant and psychrotrophic properties with the same microorganism represents substantial potential for causing spoilage of perishable milk products. Recent trends of higher pasteurization temperatures and extended refrigerated storage time of both raw and pasteurized milk and cream products exacerbates the significance of this group of microorganisms for the dairy foods industry. Copyright ©, International Association of Milk, Food and Environmental Sanitarians.
Article
低温殺菌やチーズの品質向上と安全性を確保するため,生乳ならびに低温殺菌乳の細菌叢,とくに耐熱性菌叢の分布を明らかにしようとした.生乳は,北海道のもので,4回の搾乳分を混合し,3-5°Cのバルクミルクタンクに保存されたものである.一方,低温殺菌乳は市販のもので,製造から2日間以内のものを試料とした.各細菌叢の菌数測定は,混釈平板法およびスパイラルプレーターによる表面塗抹法によった.その結果,生乳の生菌数は104-5×104ml-1の範囲であった.グラム陰性細菌数は5×102-5×104ml-1また,乳酸菌数の分布は102-105ml-1と広範囲であった.次に,耐熱性細菌はほとんどのサンプルで102ml-1であり,一部に103ml-1を越すものも見られた.生乳と市販低温殺菌乳の汚染耐熱性細菌叢はBacillusが30.7-33.5%, Microbacterium 26.9-33.7%, Micrococcaceae 17.4-23.4%,であり,他に少数であるが,Streptococcus, Lactobacillus, Actinomycetesが分離された.また,搾乳器具類の中でもミルククローの部分に,一般細菌ならびに耐熱性細菌が高い濃度で汚染されていた.これらの部分における汚染程度は,搾乳器具類の洗浄と殺菌処理の差に影響されるものと思われた.
Article
Fluorophos and colourimetric procedures for alkaline phosphatase (ALP) testing were compared using milk with raw milk additions, purified bovine ALP additions and heat treatments. Repeatability was between 0.9% and 10.1% for Fluorophos, 3.5% and 46.1% for the Aschaffenburg and Mullen (A&M) procedure and 4.4% and 8.8% for the Scharer rapid test. Linearity ( R2) using raw milk addition was 0.96 between Fluorophos and the Scharer procedure. Between the Fluorophos and the A&M procedures, R2 values were 0.98, 0.99 and 0.98 for raw milk additions, bovine ALP additions and heat treatments respectively. Fluorophos showed greater sensitivity and was both faster and simpler to perform.
Article
P. PALACIOS. J. BURGOS, L. HOZ, B. SANZ AND J.A. ORDÓÑEZ. 1991. Spore heat resistance is reduced when combined ultrasonic and heat treatments are applied. To explain this phenomenon the substances released from Bacillus stearothermophilus spores to the surrounding aqueous medium by the ultrasonic treatment (20 kHz, 120 W, 12°C, 30 min) were studied. Calcium, dipicolinic acid and a glycopeptide of 7 kDa molecular weight were detected in the ultrasonicated medium. Fatty acids, acyl glycerols and glycolipids (but no phospholipids) were also released. The decrease of heat resistance induced by the ultrasonic treatment was attributed to the release of low molecular weight substances from the spore protoplast with consequent modification of its hydration state.
Article
Raw whole milk (RM) was pasteurized with heat-pasteurized milk (TT) and thermo-sonication (US) treatments. Batch pasteurization was used for TT and 36, 72, 108 and 120 µm of the ultrasound wave intensity (24 kHz, 400 W); in addition, the heat conditions were applied together for US. Apparent protein content of US milk decreased; butter fat content was increased by US treatment. Proximal analysis showed the presence of added water in US milk and a decrease of nonfat solids, whereas pH was decreased for US milk, lactic acid was increased and density was decreased from RM to US; also, the color of US milk was whiter. The tested parameters for the TT samples were often intermediate between RM and US samples. Statistical analysis showed a significant difference (P < 0.05) among all treatments. After 16 days, US samples (4C) did not show mesophilic growth higher than 2 log; pH, acidity and color remained constant. Thermo-sonication (US) is an available method that can improve some sensorial and quality milk characteristics such as color and appearance, while at the same time, ultrasound pasteurizes the product. Minor changes in nutritional properties of milk (i.e., protein content) have been reported after US, with the advantage of extending the shelf life of the product for more than 16 days at 4C without the use of intensive heat treatments. This is a potential technology for use in research and development of new and current products in the dairy industry.
Chapter
Conventional methods of pasteurizing milk involve the use of heat regardless of treatment (batch, high temperature short time – HTST or ultra high temperature – UHT sterilization), and the quality of the milk is affected because of the use of high temperatures. Consequences of thermal treatment are a decrease in nutritional properties through the destruction of vitamins or denaturation of proteins, and sometimes the flavor of milk is undesirably changed. These changes are produced at the same time that the goal of the pasteurization process is achieved, which is to have a microbiological safe product, free of pathogenic bacteria, and to reduce the load of deteriorative microorganisms and enzymes, resulting in a product with a longer storage life.
Article
Numerous reports in the literature suggest pasteurisation failures in the dairy industry as a possible cause for an end product with a poor quality. Ultrasonication offers the dairy industry a non-thermal alternative to pasteurisation. The aim of this study was to evaluate the use of ultrasonication as an alternative to heat pasteurisation. Ultrasound was found to eliminate spoilage and potential pathogens to zero or to levels acceptable by South African and British milk legislation, even when initial inoculum loads of 5× higher than permitted were present before treatment. Viable cell counts of E. coli were reduced by 100% after 10.0 min of ultrasonication. The data obtained also showed that viable counts of Pseudomonas fluorescens were reduced by 100% after 6.0 min and Listeria monocytogenes was reduced by 99% after 10.0 min. An infra-red based apparatus was used to analyse raw and pasteurised milk after an ultrasonic treatment. Ultrasonication did not lead to decreases in the protein or lactose content of both raw and pasteurised milk. Kjeldahl nitrogen determinations confirmed that ultrasonication had no detrimental effect on the total protein or casein content of pasteurised milk. This study indicated that ultrasonication lead to an increase in the fat concentration. This was explained by the larger surface area of the fat globules after ultrasonication, which led to an increase in light scattering as observed by the MilkoScan. Alkaline phosphatase and lactoperoxidase activities were also investigated as potential indicators of an effective ultrasonic treatment. Ultrasonication was, however, found to be ineffective in deactivating both enzymes used regularly by the dairy industry as indicators of effective thermal processes.
Chapter
As discussed in previous chapters, most effects due to ultrasound arise from cavitation events, in particular, collapsing cavitation bubbles. These collapsing bubbles generate very high localized temperatures and pressure shockwaves along with micro-streaming that is associated with high shear forces. These effects can be used to accelerate the transport of substrates and reaction products to and from enzymes, and to enhance mass transfer in enzyme reactor systems, and thus improve efficiency. However, the high velocity streaming, together with the formation of hydroxy radicals and heat generation during collapsing of bubbles, may also potentially affect the biocatalyst stability, and this can be a limiting factor in combined ultrasound/enzymatic applications. Typically, enzymes can be readily denatured by slight changes in environmental conditions, including temperature, pressure, shear stress, pH and ionic strength.
Article
The increasing consumption of minimally processed chilled foods poses new risks in terms of public safety and foodborne infections. Thermal pasteurization (65-95 °C) reduces the numbers of unwanted vegetative cells of pathogenic and spoilage microorganisms in foods, extending food shelf-life, promoting food safety, and allowing the reduction and elimination of added chemical preservatives to foods. With respect to low-acid foods, microbial spores surviving pasteurization must be controlled, by using cold storage and transportation (1-8 °C), and a limited shelf-life. A review on the heat resistance of most common contaminant microbial flora in low-acid chilled foods is presented and a new strategy for the design of pasteurization processes based on the non-proteolytic psychrotrophic Clostridium botulinum spores thermal resistance will be approached.
Article
Ultrasound combined with heat treatment has yielded favorable results in the inactivation of microorganisms; however, the composition of food influences the rate of microbial inactivation. The objective of this research was to study the effect of butter fat content in milk on the inactivation of Listeria innocua and compositional parameters after thermo-sonication. Four butter fat contents in milk were evaluated at 63 °C for 30 min of sonication (Hielscher® UP400S, 400 W, 24 kHz, 120 μm amplitude). Results showed that inactivation of Listeria cells occurs first in fat free milk, and that the rate of inactivation decreases with increasing fat content. No degradation of protein content or color variation was observed after the treatments. The pH dropped to 6.22, and lactic acid content showed an increase of 0.015% after the treatment; solids-non-fat, density and freezing point decreased. During storage life, growth of mesophiles was retarded with sonication.Industrial relevanceUltrasound is an emerging technology that has shown positive effects in milk processing. Listeria monocytogenes represent one of the main foodborne pathogenic microorganisms in the food industry. Results of this research show that thermo-sonication is a viable technology capable of inactivating Listeria cells in milk and extending shelf-life without significant nutritional or physicochemical changes.
Article
Storage of UHT milk results in physico-chemical changes, which can sometimes lead to aggregation or sedimentation of milk. In this study, close attention was paid to the reactions occurring in semi-skimmed UHT milk during 6 months of storage at 4, 20 and 40 °C. Overall milk characterization revealed the development of the Maillard reaction and proteolysis which led to acidification of the milk. One hundred eighty-one peptides were identified by mass spectrometry for the freshly processed UHT milk and the milks stored 6 months at 4, 20 and 40 °C. The cleavage sites gave information concerning the possible actors of proteolysis. Plasmin, cathepsins B, D and G, elastase and proteases from Pseudomonas fluorescens B52 were thus found to be potential contributors to enzymatic proteolysis. Non-enzymatic proteolysis induced by heat-treatment and storage was also observed. Despite these modifications, milk particles (casein micelles and homogenized fat globules) did not exhibit many changes in zeta-potential, except for the last storage time at 40 °C where a decrease of the absolute value of about -3 mV was observed. The decrease in size in milks stored at 20 and 40 °C was only about 20 nm after 6 months. Similarly, storage of UHT milk showed that the higher the storage temperature the lower the heat stability and the higher the phosphate stability. Storage leads to physico-chemical changes in milk and, although some reactions such as acidification and proteolysis are known to be destabilizing, some of them are probably stabilizing to counterbalance the negative effects.
Article
Consumer demands for high-quality foods with “fresh-like” characteristics that require only a minimum amount of effort and time for preparation has led to the introduction of convenience foods preserved by mild treatments. Non-thermal methods allow the processing of foods below temperatures used during thermal pasteurisation, so flavours, essential nutrients, and vitamins undergo minimal or no changes. Foods can be non-thermally processed by irradiation, high hydrostatic pressure, antimicrobials, ultrasound, filtration, and electrical methods such as pulsed electric fields, light pulses, and oscillating magnetic fields. Due to technological developments, high pressure processing and high electric field pulse processing have received increased attention during the last decade. This paper focuses on high pressure treatment of foods, a process which is also used to create food and food ingredients with new sensory and functional properties including also physiological functionality. Effects of high pressure on chemical and sensory changes in foods are discussed.
Article
The contamination of milk by thermophilic bacteria is highly undesirable. However, the precise bacterial behaviour in milk is not been well quantified. In this study, Bacillus stearothermophilus was used as a representative thermophilic bacterium and the main milk components were used as substrates to study the growth behaviour of this species. The growth of B. stearothermophilus has been found to promote acidic conditions in both milk and lactose solution. It has been found that milk proteins sustain B. stearothermophilus in stationary phase for longer than other milk components during batch growth. Milk protein solution which contained B. stearothermophilus formed thick, dense porous lumps, more rapidly than that without bacteria. The presence of biological material on heat exchanger surfaces may promote other fouling mechanisms. Therefore, implications of the present study to the influence of the bacteria on milk fouling are also discussed.
Article
The responses of Escherichia coli K12 to sonication, manosonication (MS), thermosonication (TS), and manothermosonication (MTS) treatments were investigated at 40, 47, 54, and 61 °C and 100, 300, 400, and 500 kPa, in comparison with heat only treatments at the same temperatures. The inactivation kinetics were evaluated with five selected models and two statistical indices. Within the time frame (4 min) and temperature range used in this study, inactivation of E. coli K12 with a single lethal factor (heat or sonication) can be described by a first-order kinetic equation. For treatments with more than one lethal factor, non-linear inactivation curves were observed, which included a fast initial inactivation followed by a slow reduction in microbial survival counts. The biphasic model produced the best fit of the inactivation data compared to other non-linear models. Environmental scanning electron microscopy images showed extensive cell damage and breakage on E. coli K12 cells treated by MS, TS, and MTS.
Article
The thermophilic bacilli, such as Anoxybacillus flavithermus and Geobacillus spp., are an important group of contaminants in the dairy industry. Although these bacilli are generally not pathogenic, their presence in dairy products is an indicator of poor hygiene and high numbers are unacceptable to customers. In addition, their growth may result in milk product defects caused by the production of acids or enzymes, potentially leading to off-flavours. Dairy thermophiles are usually selected for by the conditions during dairy manufacture. These bacteria are able to grow in sections of dairy manufacturing plants where temperatures reach 40-65°C. Furthermore, because they are spore formers, they are difficult to eliminate. In addition, they exhibit a wide temperature growth range, exhibit a fast growth rate (generation time of approximately 15-20 min) and tend to readily form biofilms. Many strategies have been tested to remove, prevent and/or delay the formation of thermophilic bacilli biofilms in dairy manufacture, but with limited success. This is, in part, because little is known about the structure and composition of thermophilic bacilli biofilms in general and, more specifically, in milk processing environments. Therefore, new cleaning regimes often do not target the problem optimally. A greater understanding of the structure of thermophilic biofilms within the context of the milk processing environment and their link with spore formation is needed to develop better control measures. This review discusses the characteristics and food spoilage potential, enumeration and identification methods for the thermophilic bacilli, as well as their importance to dairy manufacture, with an emphasis on biofilm development and spore formation.
Article
In this chapter, milk will refer to bovine milk, either as full cream milk, skim-milk obtained by centrifugal separation, or standardised milk made by combining skim-milk with cream. Currently UK milk is not standardised, although both skim-milk and particularly semi-skim milk containing between 1 and 1.5 per cent fat have become more popular. Sales of semi-skim milk have increased from 4.74 million tonne in 1985 to almost 17.7 million tonne in 1990 (a 373 per cent increase). However, this increase has not occurred in other EC countries: France, 10 per cent; Germany, 5.4 per cent; and Netherlands 17.4 per cent. Other types of milk that require heat treatment are flavoured milk, reconstituted milk, filled milk, evaporated milk, milk modified in composition by demineralisation or lactose hydrolysis and protein-enriched milk produced by ultrafiltration. Milk from other species, such as goats, sheep and buffalo, may also be very important in some countries.
Article
The harmful effects on the quality and safety of dairy products caused by aerobic spore-forming isolates obtained from raw milk were characterized. Quantitative assessment showed strains of Bacillus subtilis, the Bacillus cereus group, Paenibacillus polymyxa and Bacillus amyloliquefaciens to be strongly proteolytic, along with Bacillus licheniformis, Bacillus pumilus and Lysinibacillus fusiformis to a lesser extent. Lipolytic activity could be demonstrated in strains of B. subtilis, B. pumilus and B. amyloliquefaciens. Qualitative screening for lecithinase activity also revealed that P. polymyxa strains produce this enzyme besides the B. cereus group that is well-known for causing a 'bitty cream' defect in pasteurized milk due to lecithinase activity. We found a strain of P. polymyxa to be capable of gas production during lactose fermentation. Strains belonging to the species B. amyloliquefaciens, Bacillus clausii, Lysinibacillus sphaericus, B. subtilis and P. polymyxa were able to reduce nitrate. A heat-stable cytotoxic component other than the emetic toxin was produced by strains of B. amyloliquefaciens and B. subtilis. Heat-labile cytotoxic substances were produced by strains identified as B. amyloliquefaciens, B. subtilis, B. pumilus and the B. cereus group. Variations in expression levels between strains from the same species were noticed for all tests. This study emphasizes the importance of aerobic spore-forming bacteria in raw milk as the species that are able to produce toxins and/or spoilage enzymes are all abundantly present in raw milk. Moreover, we demonstrated that some strains are capable of growing at room temperature and staying stable at refrigeration temperatures.
Article
To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable - temperature - on spore formation. A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8.5 h period at three different temperatures of 48, 55 and 60 degrees C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6-8 h. Spores formed at 55 and 60 degrees C and amounted to approx. 10-50% of the biofilm. No spores formed at 48 degrees C. The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores. This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus.
Article
This paper shows a systematic study of the 500 kHz frequency ultrasound efficiency on the microbial inactivation as a function of ultrasonic power delivered into the bacterial suspension. The inactivation of Escherichia coli IAM 12058, a Gram-negative bacterium and Streptococcus mutans JCM 5175, a Gram-positive bacterium is enhanced by increasing the ultrasonic power in the range of 1.7-12.4W and the logarithm of survival ratio decreases linearly with irradiation time, except for E. coli sonicated with the highest power level. The rate constants were estimated in the linear region of the plots representing survival ratio logarithm vs. sonication time. A better understanding of the inactivation process at 500kHz could be gained by suppressing the chemical effects with a radical scavenger. We find out that the rate constants increase with the ultrasonic power delivered into the solution and dramatically decrease by the addition of t-butanol as a radical scavenger to the bacterial suspension. For comparison, experiments were carried out at a low frequency level of 20kHz. It was found out that for the same ultrasonic power delivered into the bacterial suspension, the inactivation was slightly enhanced at 500kHz frequency. The examinations of bacterium performed with a TEM revealed lethal damages arising from the interaction of bacterial cells with the cavitational bubbles. A significant amount of empty cell envelopes as well as their cytoplasmatic content was detected. Thus, based on these new data, the mechanism of bacterial inactivation by ultrasounds at high frequency is discussed here.
Article
Propagated (free-field) ultrasonic energy at a frequency of 26 kHz was used to expose aqueous suspensions of bacteria (Escherichia coli, Staphylococcus aureus, Bacillus subtilis, and Pseudomonas aeruginosa), fungus (Trichophyton mentagrophytes), and viruses (feline herpesvirus type 1 and feline calicivirus) to evaluate the germicidal efficacy of ultrasound. There was a significant effect of time for all four bacteria, with percent killed increasing with increased duration of exposure, and a significant effect of intensity for all bacteria except E. coli, with percent killed increasing with increased intensity level. There was a significant reduction in fungal growth compared with that in the controls, with decreased growth with increased ultrasound intensity. There was a significant reduction for feline herpesvirus with intensity, but there was no apparent effect of ultrasound on feline calicivirus. These results suggest that ultrasound in the low-kilohertz frequency range is capable to some degree of inactivating certain disease agents that may reside in water. The physical mechanism of inactivation appears to be transient cavitation.
Article
A sample from a hot spring on the northern island of New Zealand contained five different thermophilic bacterial strains. One strain with peculiar properties, i.e. the formation of dark yellow colonies at 30 degrees C as well as at 70 degrees C, was further characterized. It was found to be a gram-positive, facultatively aerobic, motile Bacillus species, with terminal endospores. According to the physiologic properties the strain closely resembled B. coagulans. However, two typical characteristics were contradictory to this conclusion, namely the intense yellow pigmentation of the colonies and the range of growth temperature. The latter was found to reach from 40 to 70 degrees C, with an optimum at 60 degrees C under aerobic and at 65 degrees C under anaerobic conditions. Growth at moderate temperatures was slower than at 60 degrees C, but the final cell yields were almost equal. The strain can therefore be considered as facultatively thermophilic. The pigment, which was found to be located in the cytoplasmic membrane, was spectroscopically identified as a carotenoid. Because the characteristics of this strain did not correspond with any of the Bacillus species described thus far, we concluded, that we had isolated a novel strain, for which the name Bacillus flavothermus is proposed.
Article
The inactivation of Bacillus subtilis spores by ultrasonic treatments under static pressure (Mano-Sonication, MS) and a combined MS/heat treatment (Mano-Thermo-Sonication) was investigated. The sporicidal effect of MS treatments depended on static pressure, amplitude of ultrasonic waves and treatment temperature. At 70 degrees C, pressure increments up to 500 kPa caused progressively more inactivation. An MS treatment at 500 kPa and 117 microns of amplitude for 12 min inactivated approximately 99% of the B. subtilis spore population. Over 500 kPa, further increments in pressure did not increase the percentage of inactivation. In the range 90-150 microns, an exponential relationship was observed between the amplitude of ultrasonic waves under pressure and the number of survivors. While an MS treatment (20 kHz, 300 kPa, 70 degrees C, 12 min) at 90 microns inactivated 75% of the B. subtilis spore population, the same treatment at 150 microns inactivated 99.9% of this population. The MS treatments at temperatures higher than 70 degrees C (MTS) led to more spore inactivation. In the range 70-90 degrees C, the combination of heat with an MS treatment (20 kHz, 300 kPa, 117 microns, 6 min) had a synergistic effect on spore inactivation. The inactivating effect of ultrasound was due neither to titanium particles eroded from the sonication tip, nor to free radicals released during ultrasonic treatment. The MS treatments sensitized spores of B. subtilis to lysozyme.
Article
The effect of continuous flow high-intensity ultrasound (with and without heat generation) on alkaline phosphatase, gamma-glutamyltranspeptidase, lactoperoxidase, whey proteins (alpha-lactalbumin and beta-lactoglobulin), casein, and fat was studied in milk. Results were compared with those obtained using a conventional heating system having similar processing conditions. Hardly any effect on enzymes was observed when ultrasound was applied without heat generation. The highest denaturation of enzyme and whey proteins was found in samples subjected to ultrasound and heat. At 61, 70, and 75.5 degrees C a synergistic effect between ultrasound and heat was observed for the inactivation of alkaline phosphatase, gamma-glutamyltranspeptidase, and lactoperoxidase, respectively. A noticeable synergism between ultrasound and heat was detected for alpha-lactalbumin and beta-lactoglobulin denaturation. No changes in the casein were observed after any of the conditions assayed. As a consequence of ultrasound effects, a substantial reduction (up to 81.5%) in the size of the fat globule was observed. When 70 and 75.5 degrees C were achieved during high-intensity ultrasonic homogenization, a better particle distribution was observed as compared to that obtained at lower temperatures. This work describes the influence of continuous flow high-intensity ultrasound on important milk components as a first step for future processing applications.
Article
Alternative methods for pasteurization and sterilization are gaining importance, due to increased consumer demand for new methods of food processing that have a reduced impact on nutritional content and overall food quality. Ultrasound processing or sonication is one of the alternative technologies that has shown promise in the food industry. Sonication alone is not very effective in killing bacteria in food; however, the use of ultrasound coupled with pressure and/or heat is promising. Thermosonic (heat plus sonication), manosonic (pressure plus sonication), and manothermosonic (heat and pressure plus sonication) treatments are likely the best methods to inactivate microbes, as they are more energy-efficient and effective in killing microorganisms. Ultrasonic processing is still in its infancy and requires a great deal of future research in order to develop the technology on an industrial scale, and to more fully elucidate the effect of ultrasound on the properties of foods.
Article
Because of its food spoiling capacity on the one hand and its significant role in the production of industrially valuable products on the other, Bacillus coagulans is of economic concern. Several studies have revealed a great deal of diversity within the species and this has led to a number of taxonomic adjustments. The present study aims to clarify the diversity within Bacillus coagulans sensu stricto and determine the taxonomic status of the species. Therefore, a polyphasic study was performed on a set of B. coagulans strains from diverse habitats. Techniques as ARDRA, SDS-PAGE of whole cell proteins, FAME analysis, routine phenotypic tests and rep-PCR illustrate considerable intra-species heterogeneity, while 16S rDNA sequence comparison and DNA-DNA relatedness support the accommodation of these strains in one species. Although most techniques demonstrate appreciable heterogeneity among the Bacillus coagulans strains, the intraspecies groupings are not consistent throughout all the methods applied and are not supported by any economic, historic or practical traits. Therefore, a division in subspecies seems inappropriate. In attempt to achieve a better species delineation, an emended description of Bacillus coagulans is included.
Article
A recent example of a micro-organism causing undesired growth in consumer milk is Bacillus sporothermodurans producing highly heat-resistant spores (HRS) which may survive ultra-high temperature (UHT) treatment or industrial sterilization. Molecular typing showed a heterogeneous group of farm isolates (non-HRS strains), but a clonal group of UHT isolates from diverse European countries and other continents (HRS-clone) suggesting a common source. During a survey of Belgian dairy farms for the presence of potentially highly heat-resistant spore formers, high numbers of these spores were detected in filter cloth, green crop and fodder samples. The strain collection showed a high taxonomic diversity with 18 potentially new species and with Bacillus licheniformis and Geobacillus pallidus as predominating species overall. Seventeen B. sporothermodurans isolates were identified, mainly originating from feed concentrate. Heat resistance studies showed the UHT resistance of B. sporothermodurans spores present in industrially contaminated UHT milk, but a lower heat resistance of laboratory-grown strains (HRS and non-HRS). Hydrogen peroxide, used as sanitizer in the dairy industry, was found to induce higher heat resistance of laboratory-grown B. sporothermodurans strains to a certain level. This indicates that sublethal stress conditions may affect the heat resistance. By transmission electron microscopy, structural differences at the spore level were found between HRS and non-HRS strains. The data indicate that the attainment of extreme heat resistance is rather multifactorial.