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Short communication: Evaluation of a sol-gel–based stainless steel surface modification to reduce fouling and biofilm formation during pasteurization of milk

Open ArchivePublished:January 25, 2017DOI:https://doi.org/10.3168/jds.2016-12141

      ABSTRACT

      Milk fouling and biofilms are common problems in the dairy industry across many types of processing equipment. One way to reduce milk fouling and biofilms is to modify the characteristics of milk contact surfaces. This study examines the viability of using Thermolon (Porcelain Industries Inc., Dickson, TN), a sol-gel-based surface modification of stainless steel, during thermal processing of milk. We used stainless steel 316L (control) and sol-gel-modified coupons in this study to evaluate fouling behavior and bacterial adhesion. The surface roughness as measured by an optical profiler indicated that the control coupons had a slightly smoother finish. Contact angle measurements showed that the modified surface led to a higher water contact angle, suggesting a more hydrophobic surface. The modified surface also had a lower surface energy (32.4 ± 1.4 mN/m) than the control surface (41.36 ± 2.7 mN/m). We evaluated the susceptibility of control and modified stainless steel coupons to fouling in a benchtop plate heat exchanger. We observed a significant reduction in the amount of fouled layer on modified surfaces. We found an average fouling weight of 19.21 mg/cm2 and 0.37 mg/cm2 on the control and modified stainless steel coupons, respectively. We also examined the adhesion of Bacillus and biofilm formation, and observed that the modified stainless steel surface offered greater resistance to biofilm formation. Overall, the Thermolon-modified surface showed potential in the thermal processing of milk, offering significantly lower fouling and bacterial attachment than the control surface.

      Key words

      Short Communication

      Milk fouling and biofilms are expensive and persistent problems for the dairy industry (
      • Baier R.E.
      Surface behaviour of biomaterials: The theta surface for biocompatibility.
      ;
      • Mérian T.
      • Goddard J.M.
      Advances in nonfouling materials: Perspectives for the food industry.
      ). Fouling of milk components is a common occurrence across many types of dairy processing equipment (e.g., plates, pipes, and flow channels;
      • Sadeghinezhad E.
      • Kazi S.N.
      • Dahari M.
      • Safaei M.R.
      • Sadri R.
      • Badarudin A.
      A comprehensive review of milk fouling on heated surfaces.
      ). Previous studies have stated that the effect of fouling on the dairy industry accounts for up to 80% of total operating costs (
      • Bansal B.
      • Chen X.D.
      A critical review of milk fouling in heat exchangers.
      ). Fouling during pasteurization of milk on the stainless steel surfaces of plate heat exchangers can be classified as type A fouling, consisting of 50 to 60% proteins and 30 to 35% minerals. Fouling necessitates frequent clean-in-place, leading to increased down time and reduced production (
      • de Jong P.
      Impact and control of fouling in milk processing.
      ).
      The fouling layer on stainless steel surfaces accelerates the adhesion of bacteria and encourages the development of biofilms (
      • Simões M.
      • Simões L.C.
      • Vieira M.J.
      A review of current and emergent biofilm control strategies.
      ). The formation of biofilms during milk processing can lead to food spoilage and economic losses (
      • Bremer P.J.
      • Fillery S.
      • McQuillan A.J.
      Laboratory scale Clean-In-Place (CIP) studies on the effectiveness of different caustic and acid wash steps on the removal of dairy biofilms.
      ). The major components of biofilms include bacteria and extracellular polymeric substances produced by bacteria (
      • Flint S.H.
      • Bremer P.J.
      • Brooks J.D.
      Biofilms in dairy manufacturing plant-description, current concerns and methods of control.
      ;
      • Mittelman M.W.
      Structure and functional characteristics of bacterial biofilms in fluid processing operations.
      ). The fouled layer and extracellular polymeric substances guard the microorganisms and help them survive most of the cleaning protocols used in the dairy industry. Biofilms formed by Bacillus spp. are resistant to high stress, are very hydrophobic, and can easily attach to processing equipment (
      • Faille C.
      • Jullien C.
      • Fontaine F.
      • Bellon-Fontaine M.-N.
      • Slomianny C.
      • Benezech T.
      Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: Role of surface hydrophobicity.
      ;
      • Simões M.
      • Simões L.C.
      • Vieira M.J.
      A review of current and emergent biofilm control strategies.
      ).
      One way to control undesirable fouling and biofilms is to modify the surface properties of the processing equipment. Sol-gel surface modification converts inorganic liquid substances into a gel that can be applied on metal surfaces to improve the surface properties. Thermolon is a sol-gel-based surface modification developed from an inorganic ceramic polymer. According to its manufacturer (Porcelain Industries Inc., Dickson, TN), “It is environmentally friendly, durable, and most importantly, it has been approved by the FDA as a food contact surface (FDA 21CFR 175.300).” The objective of the present study was to evaluate the effectiveness of Thermolon surface modification on mitigating fouling and adhesion of microorganisms during the thermal processing of milk.
      Stainless steel 316L coupons (25.4mm × 25.4mm × 0.5 mm) with a 2B finished surface were provided by Stainless Supply (Monroe, NC) and used to mimic the surface of a typical plate heat exchanger. Thermolon surface modification was done by Porcelain Industries (Dickson, TN).
      We measured the contact angles of 3 liquids with known surface tension (
      • Costanzo P.
      • Giese R.
      • Van Oss C.
      Determination of the acid-base characteristics of clay mineral surfaces by contact angle measurements—implications for the adsorption of organic solutes from aqueous media.
      ): water (72.8 mN/m), 1-bromonaphthalene (44.4 mN/m), and ethylene glycol (48 mN/m) on the stainless steel control and Thermolon-modified surfaces using a static method with a FTA 1000 B Drop Shape instrument (Portsmouth, VA) at room temperature.
      Because γsTOT=γsd+γsp (where γsd and γsp are the dispersive and polar components of the solid surface energy, respectively), solid surface energy can be determined by combining Young's equation (Equation [1]) and the Owens-Wendt approach (Equation [2];
      • Santos O.
      • Nylander T.
      • Rosmaninho R.
      • Rizzo G.
      • Yiantsios S.
      • Andritsos N.
      • Karabelas A.
      • Müller-Steinhagen H.
      • Melo L.
      • Boulangé-Petermann L.
      • Gabet C.
      • Braem A.
      • Trägårdh C.
      • Paulsson M.
      Modified stainless steel surfaces targeted to reduce fouling––Surface characterization.
      ):
      γslTOT=γsTOTγlTOTcosθ,
      [1]


      where γslTOT is the total interfacial surface tension between solid and liquid, γsTOT and γlTOT are the surface tension of the solid and the liquid, respectively, and the contact angle θ; and
      γslTOT=γsTOT+γlTOT(γldγsd)122(γlpγsp)12,
      [2]


      where γld and γlp are the dispersive and polar contributions of the liquid (Table 1).
      Table 1Surface tension values (mN/m;
      • Costanzo P.
      • Giese R.
      • Van Oss C.
      Determination of the acid-base characteristics of clay mineral surfaces by contact angle measurements—implications for the adsorption of organic solutes from aqueous media.
      ;
      • Santos O.
      • Nylander T.
      • Rosmaninho R.
      • Rizzo G.
      • Yiantsios S.
      • Andritsos N.
      • Karabelas A.
      • Müller-Steinhagen H.
      • Melo L.
      • Boulangé-Petermann L.
      • Gabet C.
      • Braem A.
      • Trägårdh C.
      • Paulsson M.
      Modified stainless steel surfaces targeted to reduce fouling––Surface characterization.
      ) and contact angle for the liquids used on control and modified coupons
      Values are an average of 9 measurements (3 distinct regions on 3 independent samples) ± SE. γTOTl is the surface tension of the liquid; γdl and γpl are the dispersive and polar contributions of the liquid, respectively.
      LiquidγTOTlγdlγplContact angle (°)
      Stainless steelThermolon
      Water72.821.85182.9 ± 1.2105.5 ± 0.9
      1-Bromonaphthalene44.444.4021.7 ± 1.044.9 ± 1.5
      Ethylene glycol48291965.9 ± 1.472.4 ± 0.8
      1 Values are an average of 9 measurements (3 distinct regions on 3 independent samples) ± SE. γTOTl is the surface tension of the liquid; γdl and γpl are the dispersive and polar contributions of the liquid, respectively.
      We determined the surface roughness of different substrates using a Wyko NT1100 Optical Profiler (VEECO, Tucson, AZ). The field of view was 450 × 592 μm, and the results were reported as an average of duplicate samples, with 5 scans of each sample.
      We conducted fouling experiments in a laboratory-designed benchtop plate heat exchanger as shown in Figure 1, fitted with control and modified coupons, respectively. Different batches of raw milk were collected from the dairy plant at Kansas State University and kept at 4°C before use. Each batch of milk was divided into 2 for tests using control and Thermolon-modified stainless steel coupons. The milk inlet temperature was set at 40°C, and the hot water temperature in the second water bath was maintained at 88–90°C to maintain the milk outlet temperature at ∼85°C. Raw milk was pumped through the benchtop plate heat exchanger for 7.5 h, with a flow rate of 22 mL/min. After each test, the plate heat exchanger was dissembled, and the weight of the milk deposit on the coupons was measured after air drying for 15 min by recording the difference in weight of the clean plates versus the air-dried fouled substrates.
      Figure thumbnail gr1
      Figure 1Schematic of the benchtop plate heat exchanger (PHE) setup to simulate milk pasteurization and generate milk fouling. Color version available online.
      We carried out scanning electron microscope analysis using the Hitachi S-3500N (Tokyo, Japan). Clean stainless steel coupons (control and modified) were analyzed directly at an accelerating voltage off 20 kV. Milk fouling on the coupons was air-dried at room temperature and then coated with a 10-nm layer of 99% gold. The fouling layer was then observed at an accelerating voltage of 10 kV.
      We used aerobic spore-forming Bacillus licheniformis (ATCC 6643) to develop biofilms on control and modified stainless steel coupons at 50°C using the method described in our previous study (
      • Jindal S.
      • Anand S.
      • Huang K.
      • Goddard J.
      • Metzger L.
      • Amamcharla J.
      Evaluation of modified stainless steel surfaces targeted to reduce biofilm formation by common milk sporeformers.
      ). The biofilm embedded cells in 72 h, and matured biofilms formed on control and modified stainless steel coupons were enumerated by swabbing an area of 6.45 cm2 and plating on brain heart infusion (BHI) agar plates (
      • Jindal S.
      • Anand S.
      • Huang K.
      • Goddard J.
      • Metzger L.
      • Amamcharla J.
      Evaluation of modified stainless steel surfaces targeted to reduce biofilm formation by common milk sporeformers.
      ).
      Milk fouling and biofilm formation tests were conducted in triplicate. We calculated milk deposit weight and bacterial counts as mean values and standard deviations. We compared fouling, bacteria attachment, and surface property results using SAS software (version 9.4; SAS Institute Inc., Cary, NC) and set the least significance difference at P < 0.05.
      The properties of the milk contact surface play an important role in fouling and biofilm formation during dairy processing. We observed white, spongy-like deposit on control surfaces and less fouling on modified surfaces (Figure 2). The mean fouling weight on the control surfaces was 19.21 ± 2.25 mg/cm2, and on the modified surfaces, it was 0.37 ± 0.28 mg/cm2 (Table 2). Fouling weight was decreased up to 98% after sol-gel modification. We found more bacteria on control surfaces, at log 4.35 cfu/cm2; the modified surface showed a reduction to log 3.38 cfu/cm2 (Table 2).
      Figure thumbnail gr2
      Figure 2Representative images of fouling on (A) control and (B) Thermolon-modified stainless steel coupons after running raw milk for 7.5 h at 85°C in the benchtop plate heat exchanger.
      Table 2Surface energy, surface roughness, average weight of milk deposit, and viable counts in biofilms on control and modified coupons
      SampleSurface energy
      Surface energy values are the average of 3 independent samples ± SE.
      (mN/m)
      Surface roughness
      Surface roughness values are the average of duplicate samples with 5 scans of each sample ± SE.
      (nm)
      Average weight of milk fouling
      The values of milk fouling weight and bacterial counts in the biofilms were an average of 3 independent tests ± SE.
      (mg/cm2)
      Average counts in biofilms
      The values of milk fouling weight and bacterial counts in the biofilms were an average of 3 independent tests ± SE.
      (log10 cfu/cm2)
      Stainless steel41.36 ± 2.70
      Mean values within a column with different superscripts differ (P < 0.05).
      148.6 ± 15.0
      Mean values within a column with different superscripts differ (P < 0.05).
      19.21 ± 2.25
      Mean values within a column with different superscripts differ (P < 0.05).
      4.35 ± 0.07
      Mean values within a column with different superscripts differ (P < 0.05).
      Thermolon32.40 ± 1.40
      Mean values within a column with different superscripts differ (P < 0.05).
      199.0 ± 10.6
      Mean values within a column with different superscripts differ (P < 0.05).
      0.37 ± 0.28
      Mean values within a column with different superscripts differ (P < 0.05).
      3.38 ± 0.10
      Mean values within a column with different superscripts differ (P < 0.05).
      a,b Mean values within a column with different superscripts differ (P < 0.05).
      1 Surface energy values are the average of 3 independent samples ± SE.
      2 Surface roughness values are the average of duplicate samples with 5 scans of each sample ± SE.
      3 The values of milk fouling weight and bacterial counts in the biofilms were an average of 3 independent tests ± SE.
      When the water contact angle is over 90°, the surface is considered to be hydrophobic (
      • Bhushan B.
      • Chae Jung Y.
      Wetting study of patterned surfaces for superhydrophobicity.
      ). The water contact angle of control surfaces was slightly below 90°, and the modified surface was more hydrophobic, with a contact angle of 105° (Table 1). Early studies reported that less fouling and bacterial attachment were observed on hydrophobic surfaces (
      • Fletcher M.
      • Loeb G.
      Influence of substratum characteristics on the attachment of a marine pseudomonad to solid surfaces.
      ;
      • Pringle J.H.
      • Fletcher M.
      Influence of substratum wettability on attachment of freshwater bacteria to solid surfaces.
      ;
      • Jindal S.
      • Anand S.
      • Huang K.
      • Goddard J.
      • Metzger L.
      • Amamcharla J.
      Evaluation of modified stainless steel surfaces targeted to reduce biofilm formation by common milk sporeformers.
      ).
      The surface energy of the contact surface also affects the fouling process (
      • Sadeghinezhad E.
      • Kazi S.N.
      • Dahari M.
      • Safaei M.R.
      • Sadri R.
      • Badarudin A.
      A comprehensive review of milk fouling on heated surfaces.
      ). The relationship between fouling adhesion and material surface energy has been described by the Baier curve; substrates with surface energy 20–30 mN/m show the least atomic features of retention to protein deposition and bioadhesion (
      • Baier R.E.
      Surface behaviour of biomaterials: The theta surface for biocompatibility.
      ). In the present study, the control surface had a surface energy of 41.36 ± 2.7 mN/m, and that of the modified surface was lower, with a value of 32.40 ± 1.4 mN/m (Table 2). This finding explains the easy-release property of the Thermolon-modified surface: it has a lower surface energy, close to the bio-fouling release zone (
      • Baier R.E.
      Surface behaviour of biomaterials: The theta surface for biocompatibility.
      ).
      Scanning electron microscopy images were acquired to investigate surface morphology. As shown in Figure 3A, images of the 2B finished stainless steel surfaces revealed cracks. This might have been due to the steps involved in producing the 2B surface: an annealing oxidizing atmosphere followed by picking to remove the oxide layer formed in the previous step (
      • Frank J.F.
      • Chmielewski R.
      Influence of surface finish on the cleanability of stainless steel.
      ;
      • Jullien C.
      • Bénézech T.
      • Carpentier B.
      • Lebret V.
      • Faille C.
      Identification of surface characteristics relevant to the hygienic status of stainless steel for the food industry.
      ). The sol-gel surface modification (Figure 3B) masked all surface characteristics of the stainless steel substrate, and the scanning electron microscope image showed a more heterogeneous topography, with no cracks on the top, leading to a more uniform surface. These surface differences could have led to the differences in the fouled microstructure. On the control surfaces, we observed thick and rugged milk deposits with sublayers and porous structures (Figure 3C), which might have been due to the fouling-induced heat transfer difference across the heat exchanger surface. On Thermolon-modified surfaces, the fouling layer was thinner and smooth, with small granules (Figure 3D).
      Figure thumbnail gr3
      Figure 3Scanning electron microscope images of clean and used coupons. (A) Clean control coupon; (B) clean Thermolon-modified coupon; (C) milk fouling on control coupon; (D) milk fouling on Thermolon-modified coupon.
      Finally, the roughness of the modified surface was slightly higher (surface roughness = 199 ± 10.6 nm) than that of the control surface (surface roughness = 148.6 ± 15.0 nm; Table 2). There appears to be no direct correlation between surface roughness and bio-fouling formations (
      • Tide C.
      • Harkin S.R.
      • Geesey G.G.
      • Bremer P.J.
      • Scholz W.
      The influence of welding procedures on bacterial colonization of stainless steel weldments.
      ).
      Overall, the properties of the milk contact surface influenced the formation of milk deposit and biofilms. After Thermolon modification, the surface became more uniform and hydrophobic, with lower surface energy. Compared with the modified surface, we observed more milk fouling and bacterial attachment on the less hydrophobic control surfaces under the same conditions. These results provided evidence for the potential of using Thermolon-modified heat exchangers in dairy thermal processing to reduce milk fouling and biofilm formation, increasing process efficiency and enhancing the microbial quality of the final product.
      The durability of surface modification is an important parameter for use in food-processing applications. In future studies, we will investigate the reusability of the Thermolon surface modification.

      ACKNOWLEDGMENTS

      This work was financially supported by the National Dairy Council. The authors also acknowledge Ravindra Thakkar from Kansas State University for scanning electron microscope analysis and Qiang Ye from Kansas University for surface roughness analysis. Contribution no. 17-113-J from the Kansas Agricultural Experiment Station.

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