foods

Article One-Pot Synthesis of Derivatives from Whey Permeate

Maryam Enteshari and Sergio I. Martínez-Monteagudo * Dairy and Food Science Department, South Dakota State University, Alfred Dairy Science Hall, Brookings, SD 57007, USA; [email protected] * Correspondence: [email protected]; Tel.: +1-605-688-4118

 Received: 17 May 2020; Accepted: 5 June 2020; Published: 13 June 2020 

Abstract: The simultaneous production of lactulose (LAU), lactobionic (LBA), and organic from sweet and acid whey permeate (SWP and AWP) via catalytic synthesis (5% Ru/C) was studied in a continuous stirred-tank reactor. At selected conditions (60 ◦C, 60 bar, and 600 rpm), a maximum conversion of lactose (37 and 34%) was obtained after 90 min for SWP and AWP, respectively. The highest yield calculated with respect to the initial concentration of lactose for LAU was 22.98 0.81 ± and 15.29 0.81% after only 30 min for SWP, and AWP, respectively. For LBA, a maximum yield was ± found in SWP (5.23%) after 210 min, while about 2.2% was found in AWP. Six major organic acids (gluconic, pyruvic, lactic, formic, acetic, and citric acid) were quantified during the one-pot synthesis of lactose.

Keywords: one-pot synthesis; catalysis; lactulose; lactobionic acid

1. Introduction Whey is the main byproduct obtained from the manufacture of cheese, yogurt, and milk protein concentrates [1]. It is the yellowish liquid separated from curds, and it is mainly made of water (~94%), lactose (~5%), proteins (~1%), minerals (~1%), and milk fat (~0.5%) [2]. As a byproduct, whey is concentrated and further fractionated to produce a wide array of products and ingredients with food and pharmaceutical applications. Examples of ingredients derived from whey are milk serum protein concentrate, whey powder, whey protein concentrate, whey protein hydrolysate, and whey protein isolate. Manufacturing details on the production of milk protein ingredients can be found elsewhere [3,4]. Over the last few years, the number of food formulations containing milk proteins has considerably increased due to the functional and nutritional properties of milk proteins [5]. Nowadays, products such as sport drinks, desserts, beverages, snacks, dietary, and weight management supplements are formulated with milk proteins. Overall, the manufacture of concentrates of milk proteins involves numerous unit operations, such as thermal treatment, selective fractionation, concentration, and drying. Selective fractionation by means of membrane technology is the key processing step in which the protein fraction is sequentially separated from the whey stream. The separation is performed by multiple filtration stages, where the proteins are concentrated in the retentate, while lactose and minerals are concentrated in permeate stream. The byproduct derived from the production of milk proteins is known as whey permeate, and it contains a considerable amount of lactose (75–80% on dry basis). Lactose (LA) presents technological challenges, such as poor solubility and low sweetness index, as well as malabsorption by a certain population, which has limited its further utilization [6]. Consequently, there is a considerable industrial interest to further utilize LA as a feedstock for the production of lactose-based ingredients [7,8]. As a feedstock, lactose has the potential to undergo

Foods 2020, 9, 784; doi:10.3390/foods9060784 www.mdpi.com/journal/foods Foods 2020, 9, x FOR PEER REVIEW 2 of 11 Foods 2020, 9, 784 2 of 11 production of lactose-based ingredients [7,8]. As a feedstock, lactose has the potential to undergo differentdifferent reactions reactions leading leading to the formation of value-addedvalue-added compounds. Figure Figure 1 1 illustratesillustrates the the simultaneoussimultaneous formation formation of of lactulose lactulose (LAU) (LAU) and and lactobionic lactobionic acid acid (LB (LBA),A), two two of of the the most most versatile versatile lactoselactose-derived-derived ingredients ingredients,, with with applications applications in in food, food, dairy, dairy, and pharmaceutical formulations.

FigureFigure 1 1.. ReactionReaction pathway pathway for for the the formation formation of of lactulose lactulose and and lactobionic lactobionic acid acid via via isomerization isomerization and and dehydrogenation,dehydrogenation, respective respectively.ly.

β d d LactuloseLactulose (4 (4--OO--β-D- --galactopyranosyl-galactopyranosyl-D--fructofuranose),fructofuranose), an an isomer isomer of of LA, LA, is is a a disaccharide disaccharide mademade of of galactose and and fructose. fructose. It It is is used used in in the the treatment treatment of of hepatic hepatic encephalopathy encephalopathy and and chronic chronic constipationconstipation [9]. [9]. Industrially, Industrially, LAU LAU is is synthesized synthesized by by either either hom homogenousogenous catalysis catalysis under under alkaline alkaline conditionsconditions,, or or by by enzymatic synthesis using glycosyltransferases and glycosidases [[10].10]. Reaction Reaction schemesschemes and and conditions conditions for for LAU LAU synthesis synthesis can can be be found found elsewhere elsewhere [11]. [11 On]. Onthe theother other hand, hand, LBA LBA (4- β d O(4--βO-galactopyranosyl- -galactopyranosyl--D-gluconi-gluconicc acid) acid) is isan an aldonic aldonic acid acid or or sugar sugar-acid-acid made made up up of of galactose galactose linked linked toto a a gluconic acid [12]. [12]. The The LBA LBA molecule molecule has has various various functionalities functionalities,, such such as as antioxidant, antioxidant, chelating, chelating, andand humectant humectant,, arising arising from from the the high high number number of of hydroxyl hydroxyl groups. groups. The The partial partial oxidatio oxidationn of of LA LA results results inin the the formation formation of of LBA, LBA, where where the the aldehyde aldehyde group group is is converted converted into into its its corresponding corresponding carbonyl carbonyl groupgroup via via selective selective oxidation oxidation [13]. [13]. Most Most of ofthe the LBA LBA commercialized commercialized in inthe the world world is produced is produced via via1) biocatalytic1) biocatalytic conversion conversion using using dehydrogenase dehydrogenase or or oxidoreductase, oxidoreductase, or or 2) 2) heterogeneous heterogeneous catalysis catalysis using using Pd,Pd, Pt, Pt, or or Ru Ru as as insoluble insoluble catalysts. catalysts. The The m manufacturinganufacturing methods methods of of LBA LBA can can be be found found elsewhere elsewhere [12]. [12]. OneOne common common strategy employedemployed inin the the industrial industrial production production of of lactose-derived lactose-derived ingredients ingredients is the is theuse use of purifiedof purified lactose lactose (monohydrate (monohydrate or pharmaceuticalor pharmaceutical grade) grade) as aas feedstock, a feedstock, which which minimizes minimizes the theformation formation of secondary of secondary products. products. Fundamentally, Fundamentally, such a strategysuch a strategy aims to produce aims to a produce single compound a single compoundvia multi-step via multi flow,- involvingstep flow, theinvolving formation, the formation, separation, separation, and isolation and isola of secondarytion of secondary and primary and primaryproducts. products. Alternatively, Alternatively, the conversion the conversion of lactose of through lactose one-pot through synthesis one-pot representssynthesis represents an innovative an innovativeutilization of utilization lactose. One-pot of lactose. conversion One-pot involves conversion the synthesis involves of a mixture the synthesis of compounds of a mixture that can of be compoundsused with minimal that can separation be used in with the final minimal formulation. separation This in has the been final exemplified formulation. by our This group, has where been exemplifieda sweetening by syrup our group, from aqueous where a lactose sweetening was produced syrup from via aqueous one-pot synthesislactose was (enzymatic produced hydrolysis via one- potfollowed synthesis by catalytic(enzymatic isomerization) hydrolysis followed [14,15]. Similarly,by catalytic Zaccheria isomerization) [16] produced [14,15]. aSimilarly, mixture ofZaccheria sorbitol [16]and dulcitol produced from a lactosemixture via of one-pot sorbitol catalytic and dulcitol conversion. from Gallezot lactose [ via17] conceptualized one-pot catalytic and conversion. exemplified Gallezotthe production [17] conceptualized of a pool of molecules and exemplified from biomass the production via one-pot of a catalytic pool of synthesis.molecules Underfrom biomass the one-pot via oneapproach,-pot catalytic the high synthesis. concentration Under ofthe lactose one-pot present approach, in whey the permeatehigh conce canntration be used of lactose as an inexpensive present in wheyfeedstock permeate for a varietycan be used of chemical as an inexpensive modifications, feedstock such as for hydrogenation, a variety of chemical isomerization, modifications and oxidation., such as hydrogenation,During the catalytic isomerization, oxidation and of lactose,oxidation. the first products formed are LBA and LAU, followed by 2-keto-lactobionicDuring the catalytic acid as oxidation a secondary of lactose, product the [18 first]. Preliminary products formed results are showed LBA and that LAU, increasing followed the byoxygen 2-keto pressure-lactobionic improved acid as the a yieldsecondary of LAU product during [18]. the oxidationPreliminary of lactoseresults overshowed Ru/ C[that19 ].increasing It should thebe highlightedoxygen pressure that the improved production the yield of LAU of andLAU LBA during was the evaluated oxidation using of lactose lactose over monohydrate Ru/C [19]. as It a shouldfeedstock. be Nevertheless, highlighted these that resultsthe production present an of opportunity LAU and for LBA the simultaneous was evaluated production using lactoseof LAU monohydrateand LBA directly as a feedstock. from whey Nevertheless, permeate. This these work results aimed present at producing an opportunity LAU and for LBAthe simultaneous via a one-pot productioncatalytic oxidation of LAU fromand LBA sweet directly and acid from whey whey permeate. permeate. This work aimed at producing LAU and LBA via a one-pot catalytic oxidation from sweet and acid whey permeate.

Foods 2020, 9, 784 3 of 11

2. Materials and Methods

2.1. Materials Firstly, α-d-Lactose monohydrate (98%, Acros Organics, Fair Lawn, NJ, USA), lactulose (99%, Alfa Aesar, Haverhill, MA, USA), lactobionic acid (97%, Sigma-Aldrich, St. Louis, MO, USA), D-(+)-glucose (99%, Sigma-Aldrich), D-(+)-galactose (99%, Sigma-Aldrich), D-gluconic acid (99%, Sigma-Aldrich), pyruvic acid (98%, Sigma-Aldrich), L-(+)-lactic acid (85%, Sigma-Aldrich), formic acid (99%, Fisher Scientific, Waltham, MA, USA), citric acid (99%, Sigma-Aldrich), and orotic acid (98%, Sigma-Aldrich) were purchased from commercial suppliers. Reduced ruthenium supported on activated carbon (5% Ru/C, Alfa Aesar) was purchased from commercial suppliers, and it was used without further preparation. Sweet whey permeate (SWP) was obtained from a regional cheese factory (Valley Queen Co., Milbank, SD, USA), while the acid whey permeate (AWP) was obtained from a Greek yogurt plant (Chobani Co., Twin Falls, ID, USA).

2.2. Preparation of Samples A 10% wt./wt. solution of lactose (LAS) was prepared by dissolving D-lactose monohydrate in distilled water. Samples of SWP and AWP were analyzed for pH, total protein, fat content, total solids, and total non-volatile solids. The pH was measured in 10 mL of the sample using a triode epoxy electrode (Orion Versa Star Pro, Thermo Fisher Scientific). The protein content was determined by the Kjeldahl method, while the fat content was measured using the Mojonnier fat extraction, following the guidelines of the Association of Official Analytical Chemists (AOAC) methods 989.05 and 991.20, respectively [20]. Total solids (TS) and total non-volatile solids (TNVS) were determined using the AOAC method 990.20 [20]. Table1 shows the compositional characteristics of SWP and AWP.

Table 1. Physicochemical characteristics of sweet whey permeate and acid whey permeate.

Parameters Sweet Whey Permeate Acid Whey Permeate pH 6.23 0.01 4.38 0.02 ± ± Total solids (g/L) 87.38 0.25 85.35 0.06 ± ± Total non-volatile solids (g/L) 8.23 0.17 9.22 0.16 ± ± Fat (g/L) 0.85 0.23 1.81 0.10 ± ± Total protein (g/L) 3.02 0.01 3.95 0.08 ± ± Lactose (g/L) 76.42 4.3 61.73 6.29 ± ± Organic acids (g/L) 7.11 0.35 17.91 0.89 ± ± Mean standard deviation (n = 3). ±

2.3. Experimental Treatments A set of experiments was first conducted to evaluate the influence of pressure (15, 40, 60, and 80 bar) and temperature (50, 60, 70, and 80 ◦C) on the conversion rate of lactose in a 10% wt./wt. solution of lactose. Initial rates (ro) of lactose conversion were calculated using Equation (1):

d[La] ro = (1) dt t=0

At selected conditions, the second set of experiments was conducted to evaluate the formation of LAU and LBA directly from sweet whey permeate (SWP), and acid whey permeate (AWP).

2.4. One-Pot Synthesis The one-pot conversion of LA was carried out in a continuous stirred-tank reactor (BR-300, Berghof Products & Instruments, Berghof, Germany). Figure2 schematically represents the one-pot conversion of lactose directly from whey permeate. Features and characteristics of the reactor, as well as the Foods 2020, 9, 784 4 of 11 Foods 2020, 9, x FOR PEER REVIEW 4 of 11 workingas the working conditions, conditions, have been have explained been explained thoroughly thoroughly in previous in previous work [work21]. Briefly, [21]. Briefly, the reactor the reactor vessel wasvessel loaded was loaded with 400 with mL 400 of mL either of LAS,either SWP,LAS, orSWP, AWP, or containingAWP, containing 0.5 g/L 0.5 of Rug/L/ Cof catalyst. Ru/C catalyst. Then, theThen, vessel the vessel was lidded, was lidded, clamped, clamped, and heated and heated up to theup targetto the temperature.target temperature. During During heating, heating, the vessel the wasvessel pressurized was pressurized with compressed with compres airsed as air an as oxidizing an oxidizing agent agent (99.99% (99.99% purity, purity, Praxair, Praxair, Sioux Sioux Falls, Falls, SD, USA).SD). The The initial initial time time of of the the catalytic catalytic reaction reaction was was considered considered when when the the solution solution reached reached the target temperature and pressure. Once the working conditions were obtained, the reaction was mo monitorednitored over 210 min, withdrawing samples (~15(~15 mL) every 30 min and stored at −2020 °CC until until further further analysis. analysis. − ◦ Samples were withdrawn from the sampling port ((6) in Figure2 2),), consisting consisting of of aa setset ofof needleneedle valvesvalves that allowed the subtraction of samplessamples withoutwithout losinglosing pressurepressure insideinside thethe reactor.reactor.

Figure 2. Schematic of the continuous stirred-tank reactor used for the one-pot conversion of lactose Figure 2. Schematic of the continuous stirred-tank reactor used for the one-pot conversion of lactose permeate: (1) data logger, (2) stirrer, (3) tachometer, (4) pressure gauge, (5) thermocouple, (6) sampling permeate: (1) data logger, (2) stirrer, (3) tachometer, (4) pressure gauge, (5) thermocouple, (6) port, (7) cooling system, (8) nitrogen cylinder, (9) stainless-steel reactor, (10) electrical heater. sampling port, (7) cooling system, (8) nitrogen cylinder, (9) stainless-steel reactor, (10) electrical 2.5. Quantificationheater. of Reaction Products The reaction products derived were quantified by liquid chromatography-mass spectrophotometry 2.5. Quantification of Reaction Products (LC-MS). Firstly, samples withdrawn from the reactor were filtered through a 20-25 µm filter (NO 541 HardenedThe Ashless, reaction and products 110 mm diameter, derived WhatmanTM were quantified Co., Marlborough, by liquid MA,chromatography USA) to remove-m theass spentspectrophotometry catalyst. Then, (LC the-MS). filtered Firstly, solution samples was withdrawn diluted 10-fold from withthe reactor HPLC were grade filtered water, through followed a 20 by- centrifugation25 μm filter (NO using 541 ultracentrifugationHardened Ashless, filters and 110 (Amicon mm diameter,® filters, WhatmanTM Merc Millipore, Co., Billerica, Marlborough, MA, USA) MA, at 9000USA) rpm to remove for 10 min the (accuSpinspent catalyst. Micro Then, 17R, the Fisher filtered Scientific, solution Waltham, was diluted MA, USA). 10-fold After with centrifugation, HPLC grade thewater, precipitate followed was by discarded,centrifugation while using the supernatantultracentrifugation was used filters for analysis. (Amicon An® filters, aliquot Merc of 10 Millipore,µL from theBillerica, supernatant MA, USA) was injectedat 9000 rpm into afor Shimadzu 10 min (accuSpin LC system Micro (LC-20AD, 17R, Fisher Shimadzu Scientific Corp,, Waltham, Kyoto, Japan), MA, combinedUSA). After with centrifugation, a Qtrap 5500 triple the precipitate quadrupole was mass discarded spectrometer, while (AB the Sciex, supernatant Foster City, was CA, used USA). for Analytesanalysis. wereAn aliquot eluded of by 10 means µL from of an the HPX-87C supernatant column was (250x4.0 injected mm, into Bio-Rad a Shimadzu Aminex, LC Hercules, system (LC CA,- USA)20AD, operated Shimadzu at 80 Corp,◦C with Kyoto, a mobile Japan) phase, combined consisted with of acetonitrile: a Qtrap water5500 triple (20:80 quadrupolev/v), at a flow mass rate ofspectrometer 0.2 mL/min. (AB Mass Sciex, spectrophotometer Foster City, CA, analysis USA). Ana waslytes operated were in eluded negative by mode means at of a antemperature HPX-87C ofcolumn 500 ◦ C,(250x4.0 a curtain mm, gas Bio of-Rad 30 psi, Aminex, an Hercules, source gas CA, of USA) 15 psi operated for nebulizer at 80 (GS1),°C with and a mobile heater (GS2).phase Samplesconsisted were of acetonitrile: quantified accordingwater (20:80 to HPLC-gradev/v), at a flow analytical rate of standards.0.2 mL/min. Mass spectrophotometer analysis was operated in negative mode at a temperature of 500 °C , a curtain gas of 30 psi, an ion

Foods 2020, 9, 784 5 of 11

2.6. Quantification of Organic Acids The organic acids formed during the catalytic oxidation of LA were analyzed by HPLC, according to the methodology reported by Zeppa et al. [22], with some modifications. An HPLC instrument (Beckman Coulter, Inc., Fullerton, CA, USA) equipped with a solvent delivery module (System Gold® 125), a multichannel wavelength scanning detector (190–600 nm, System Gold 168 detector), and a refractive index detector (RI-2031, Jasco Corporation, Hachioji, Japan) were used for the analysis. The separation of organic acids was performed using an ion exclusion column (ROA-Organic Acid H+ 8%, Phenomenex, Torrance, CA, USA) heated at 60 ◦C. The mobile phase was a sulfuric acid solution 1 (0.013 N), at a flow rate of 0.5 mL min− .

2.7. Data Analysis Lactose conversion was expressed as a percentage of converted LA into its derivatives, according to Equation (2). [LAo] [LAt] CLA = − 100 (2) [LAo] × where CLA—conversion of lactose (%), [LAo]—initial concentration of lactose (mM), and [LAt]— concentration of lactose at a given reaction time. The formation of LAU and LBA was determined as the product yield (Yi) according to Equation (3).

Concentration o f target product Y = 100 (3) i Initial concentration o f lactose ×

Experimental runs were conducted in duplicate, and all the figures were made with SigmaPlot software V11 for Windows (Systat Software, Inc., Chicago, IL, USA).

3. Results and Discussion

3.1. Reaction Conditions Figure3 shows a graphical representation of the temperature-pressure history during the conversion of LA. Each experimental treatment started with the heading time, which includes loading, pressurization (arrow (1)), and heating of the vessel. Start of the reaction time (arrow (2)) was considered when both the target temperature and pressure were achieved. Afterward, samples (~15 mL) were withdrawn for analysis at interval times of 30, 90, 150, and 210 min. The end of the reaction time (arrow (3)) marked the beginning of the cooling and depressurization (arrow (4)). Similar characterization of the temperature-pressure history was reported elsewhere [21]. A detailed description of the main variables involved during the one-pot conversion allows one to meaningfully interpret the formation of reaction products, and facilitates comparison with the literature.

3.2. Initial Rates The initial rate of LA conversion is shown in Figure4. Pressure influence on the initial rates was evaluated at a constant temperature (60 ◦C, Figure4a), where the initial rates increased with the pressure until it reached a maximum value of 1.95 0.09 mmol/min at 60 bar, while a further increment ± of pressure resulted in lower values of initial rates (0.70 0.06 mmol/min at 80 bar). This finding is ± important from an economic and operational perspective, since the application of high pressure will increase the operational costs and be difficult to operate. Influence of temperature on the initial rates of lactose conversion was evaluated at a constant pressure (60 bar, Figure4b). The highest values of initial rates were obtained at 60 ◦C, and further elevation of the temperature did not significantly increase the initial rates. Overall, the magnitude of the initial rates was higher by increasing the temperature than by the increment of the pressure. This observation is in agreement with the Arrhenius and Eyring theory [23]. Foods 2020, 9, x FOR PEER REVIEW 6 of 11 Foods 2020, 9, x FOR PEER REVIEW 6 of 11 increasing the temperature than by the increment of the pressure. This observation is in agreement increasing the temperature than by the increment of the pressure. This observation is in agreement withFoods 2020the ,Arrhenius9, 784 and Eyring theory [23]. 6 of 11 with the Arrhenius and Eyring theory [23].

Figure 3. Representative temperature-pressure history during the one-pot conversion of lactose. Figure 3. Representative temperature-pressure history during the one-pot conversion of lactose. ReactionFigure 3. temperature Representative was temperature60 °C and pressure-pressure was history 60 bar. during theating — theheating one-pot time conversion (min), and of t reaction lactose.— Reaction temperature was 60 ◦C and pressure was 60 bar. theating—heating time (min), and treaction— reactionReaction time temperature (min). Arrows was 60 (1 –°C4) andrepresent pressure the startwas 60of pressurizationbar. theating—heating, the beginning time (min), of andthe reactiontreaction— reaction time (min). Arrows (1–4) represent the start of pressurization, the beginning of the reaction time,reaction the timeend of(min). the reaction Arrows time, (1–4) and represent the depressurization, the start of pressurization respectively., the beginning of the reaction time, the end of the reaction time, and the depressurization, respectively. time, the end of the reaction time, and the depressurization, respectively.

FigureFigure 4. 4. InitialInitial rate rate of lactose of lactose conversion: conversion: (a) pressure (a) pressure effect(temperature effect (temperature= 60 ◦C) and = 60 (b )°C temperature) and (b) eFigureffect (pressure 4. Initial= rate60 bar). of lactose Mean conversion:standard deviation (a) pressure within effect treatments (temperature with di =ff erent 60 °C letters) and are (b) temperature effect (pressure = 60 bar).± Mean ± standard deviation within treatments with different letters(temperaturea–c) significantly are (a– ceffect) significantly di (pressurefferent ( pdifferent =< 600.05) bar). according(p Mean< 0.05) ± according tosta Tukey’sndard todeviation test. Tukey’s within test. treatments with different letters are (a–c) significantly different (p < 0.05) according to Tukey’s test. 3.3. Conversion and Yield 3.3. Conversion and Yield 3.3. ConversionFigure5 shows and Yield the kinetic curves of lactose conversion for SWP and AWP. During the first Figure 5 shows the kinetic curves of lactose conversion for SWP and AWP. During the first 30 30 min of reaction, about 30 and 24% of lactose was converted for SWP and AWP, respectively. min ofFigure reaction, 5 shows about the 30 kinetic and 24% curves of lactose of lactose was conversionconverted forfor SWPSWP andand AWP,AWP. respectively.During the first After 30 After 210 min, the conversion of lactose reached a maximum value of about 37 and 34% for SWP 210min min, of reaction, the conversion about 30of lactoseand 24% reached of lactose a maximum was converted value of for about SWP 37 and and AWP, 34% for respectively. SWP and AWP, After and AWP, respectively. Mäki-Arvela et al. [24] reported conversion values of about 90% during the respectively.210 min, the conversionMäki-Arvela of et lactose al. [24] reached reported a maximum conversion value values of ofabout about 37 90%and during34% for the SWP oxidation and AWP, of oxidation of LA (0.86 M) under alkaline conditions (pH = 8), using Pd/C as a catalyst. The difference LArespectively. (0.86 M) Mäki under-Arvela alkaline et al. conditions [24] reported (pH conversion= 8), using values Pd/C of as about a catalyst. 90% during The difference the oxidation in the of in the conversion values can be explained by the deactivation of the catalyst and the pH of the conversionLA (0.86 M) values under can alkaline be explained conditions by the (pH deactivation = 8), using of Pd/C the catalyst as a catalyst. and the The pH difference of the solution. in the solution. Indeed, Mäki-Arvela et al. [25] reported that Ru/C rapidly underwent deactivation during Indeed,conversion Mäki values-Arvela can et be al. explained [25] reported by the that deactivation Ru/C rapidly of the underwent catalyst and deactivation the pH of the during solution. the the oxidation of lactose monohydrate solution (0.86 M), achieving conversion values of 28–32%. In oxidationIndeed, Mäki of lactose-Arvela monohydrate et al. [25] solution reported (0.86 that M), Ru/C achieving rapidly conversion underwent values deactivation of 28–32%. during In SWP the SWP and AWP, the presence of minerals and residual proteins seems to impact the activity of the andoxidation AWP, ofthe lactose presence monohydrate of minerals solution and residual (0.86 M),proteins achieving seems conversion to impact thevalues activity of 28 of–32%. the catalyst In SWP catalystand AWP, negatively. the presence However, of minerals the deactivation and residual of proteins Ru/C due seems to minerals to impact and the residualactivity of protein the catalyst needs further experimental evidence.

Foods 2020, 9, x FOR PEER REVIEW 7 of 11 negatively. However, the deactivation of Ru/C due to minerals and residual protein needs further experimental evidence. Figure 5a,b showed the formation curves for LAU and LBA corresponding to SWP and AWP, respectively. In untreated samples of SWP and AWP, the concentration of LAU and LBA was below the detection limit. An increment in the yield values of LAU was observed within the first 30 min of reaction, 22.99 ± 0.81 and 17.29 ± 0.96% for SWP and AWP, respectively. The relatively high yield values for SWP is not surprising, since the pH of the SWP (6.23 ± 0.01) favored the formation of LAU. Seo et al. [26] reported yield values of LAU of 29% (90 °C and 20 min of reaction) during the isomerization of cheese whey, using Na2CO3 (0.5%) as a catalyst. As the reaction proceeded, the yield values decreased to 14.13 ± 0.13 and 10.93 ± 0.07% for SWP and AWP, respectively. The observed reduction in the yield of LAU by increasing the reaction time is due to the hydrolysis of LAU and subsequent formation of organic acids. The mechanism of lactose isomerization consisted of epimerization and aldose–ketose interconversion. Such a network of reactions is known as Lobry de Bruyn-van Ekenstein transformation. Hajek et al. [27] have studied the isomerization of lactose in an Foodsalkaline2020 ,environment.9, 784 7 of 11

Figure 5. a Figure 5. ConversionConversion of lactose lactose and yield of lactulose lactulose and lactobionic acid over time in: ( (a) sweet sweet whey whey permeate (SWP), and (b) acid whey permeate (AWP). Temperature = 60 C and pressure = 60 bar, permeate (SWP), and (b) acid whey permeate (AWP). Temperature = 60 °C and◦ pressure = 60 bar, and and stirring rate 600 rpm. stirring rate 600 rpm. Figure5a,b showed the formation curves for LAU and LBA corresponding to SWP and AWP, In the case of LBA, the yield for the SWP (Figure 5a) increased asymptotically with the reaction respectively. In untreated samples of SWP and AWP, the concentration of LAU and LBA was below time, reaching a value of 5.23 ± 0.02%. Similar behavior, but less pronounced, was observed in the the detection limit. An increment in the yield values of LAU was observed within the first 30 min yield values of LBA for AWP (2.15 ± 0.15%). The relatively high yield of LBA for SWP, nearly 2-fold of reaction, 22.99 0.81 and 17.29 0.96% for SWP and AWP, respectively. The relatively high than AWP, is not surprising± , since the± pH of SWP (6.23 ± 0.01, Table 1) favored LBA formation. It is yield values for SWP is not surprising, since the pH of the SWP (6.23 0.01) favored the formation thought that acidic conditions hinder the interaction between molecular± oxygen and LA moieties, of LAU. Seo et al. [26] reported yield values of LAU of 29% (90 C and 20 min of reaction) during affecting LBA formation. The role of pH during the catalytic oxidation◦ of LA has been discussed the isomerization of cheese whey, using Na CO (0.5%) as a catalyst. As the reaction proceeded, elsewhere [28,29]. Mechanistically, the oxidation2 of3 aldehydes is consensually thought to occur in two the yield values decreased to 14.13 0.13 and 10.93 0.07% for SWP and AWP, respectively. The steps, hydration of the aldehyde ± group and subsequent± dehydrogenation to produce their observed reduction in the yield of LAU by increasing the reaction time is due to the hydrolysis of corresponding acid [30]. LAU and subsequent formation of organic acids. The mechanism of lactose isomerization consisted of epimerization and aldose–ketose interconversion. Such a network of reactions is known as Lobry de 3.4. Formation of Glucose, Galactose, and Organic Acids Bruyn-van Ekenstein transformation. Hajek et al. [27] have studied the isomerization of lactose in an alkalineThe environment.conversion values of lactose, as well as the yield values of LAU and LBA (Figure 5), suggest the formationIn the case of ofother LBA, products the yield derived for the from SWP hydrolysis, (Figure5a) oxidation, increased asymptoticallyand degradation. with These the products reaction time,were reachingdivided ainto value three of 5.23groups0.02%.—monosaccharides, Similar behavior, sugar but less acids, pronounced, and organic was acids observed (Figure in the 6). yield The ± valuesfirst group of LBA of compounds for AWP (2.15 is monosacc0.15%). Theharides relatively (glucose high and yield galactose) of LBA that for SWP, are formed nearly 2-folddue to than the ± AWP,hydrolysis is not of surprising, lactose, breakage since the pH of the of SWP glycosidic (6.23 linkage.0.01, Table The1) favored initial concentration LBA formation. of It glucose is thought and ± thatgalactose acidic was conditions higher hinderin AWP the (about interaction 21 and between 78 mmol/L, molecular respectively) oxygen and than LA that moieties, of SWP aff (aboutecting LBA4.61 formation.and 2.21 mmol/L, The role respectively). of pH during Acid the catalyticwhey permeate oxidation is ofthe LA byproduct has been of discussed yogurt manufacture elsewhere [28 [,3129],]. Mechanistically,and it is characterized the oxidation by a relatively of aldehydes high is concentration consensually thoughtof organic to occuracids, inTable two steps,1. As hydrationthe reaction of the aldehyde group and subsequent dehydrogenation to produce their corresponding acid [30].

3.4. Formation of Glucose, Galactose, and Organic Acids The conversion values of lactose, as well as the yield values of LAU and LBA (Figure5), suggest the formation of other products derived from hydrolysis, oxidation, and degradation. These products were divided into three groups—monosaccharides, sugar acids, and organic acids (Figure6). The first group of compounds is monosaccharides (glucose and galactose) that are formed due to the hydrolysis of lactose, breakage of the glycosidic linkage. The initial concentration of glucose and galactose was higher in AWP (about 21 and 78 mmol/L, respectively) than that of SWP (about 4.61 and 2.21 mmol/L, respectively). Acid whey permeate is the byproduct of yogurt manufacture [31], and it is characterized by a relatively high concentration of organic acids, Table1. As the reaction proceeded, the glucose in SWP (Figure6a) gradually increased with time, reaching a maximum of about 13 mmol /L after 90 min. Subsequent progression of the reaction resulted in a lower concentration of glucose (about 9 mmol/L after 210 min). A similar trend was observed for galactose in SWP (Figure6a), where a maximum Foods 2020, 9, x FOR PEER REVIEW 8 of 11

proceeded, the glucose in SWP (Figure 6a) gradually increased with time, reaching a maximum of about 13 mmol/L after 90 min. Subsequent progression of the reaction resulted in a lower concentration of glucose (about 9 mmol/L after 210 min). A similar trend was observed for galactose Foodsin SWP2020, 9 (,Figure 784 6a), where a maximum concentration (24 mmol/L) was obtained after 908 of min, 11 followed by a reduction in the concentration with the progression of the reaction. On the other hand, concentrationthe concentration (24 mmol of glucose/L) was and obtained galactose after in AWP 90 min, (Figure followed 6b) bygradually a reduction decreased in the with concentration time from withabout the 21 progression and 78 mmol/L of the reaction.at the beginning On the otherof the hand, reaction the concentration to 16 and 42 ofmmol/L glucose after and 210 galactose min, infor AWPglucose (Figure and6 galactose,b) gradually respectively. decreased It with appears time fromthat lactose about 21underwent and 78 mmol hydrolysis/L at the to beginningproduce glucose of the reactionand galactose to 16 andthat 42further mmol oxidized/L after 210to form min, their for glucose respectiv ande sugar galactose, acids, respectively.gluconic and Itgalactonic appears thatacid. lactose underwent hydrolysis to produce glucose and galactose that further oxidized to form their respective sugar acids, gluconic and galactonic acid.

FigureFigure 6. 6.Formation Formation of of organic organic acids acids over over time time in: in: ((aa)) sweet sweet whey whey permeate permeate (SWP), (SWP), and and ( b(b)) acid acid whey whey permeatepermeate (AWP). (AWP). Temperature Temperature= =60 60˚C◦C andand pressurepressure == 60 bar, andand stirringstirring raterate 600600 rpm.rpm.

TheThe sugar sugar acids acids represent represent the the second second group group of of components components derived derived from from the the catalytic catalytic conversion conversion ofof lactose. lactose. InIn SWP,SWP, the the concentration concentration of of gluconic gluconic acid acid increased increased withwith timetime untiluntil itit reached reached a a plateau plateau atat 150150 minmin (60(60 mmolmmol/L)./L). Afterward, the the concentration concentration of of gluconic gluconic acid acid marginally marginally increased increased up up to to66 66mmol/L mmol/ Lafterafter 210 210 min. min. In In the the case case of of AWP, AWP, the the concentration of gluconic acidacid reachedreached aa maximummaximum valuevalue of of 65 65 mmol mmol/L/L only only after after 30 30 min, min, followed followed by by a a reduction reduction to to about about 3030 mmolmmol/L./L. Chemical Chemical process process forfor the the production production of of gluconic gluconic acid acid consisted consisted of of catalytic catalytic oxidation oxidation of of a a concentrated concentrated glucose glucose solution solution underunder alkaline alkaline conditions conditions [ 32[32].]. TheThe thirdthird groupgroup of components under under consideration consideration corresponds corresponds to to organic organic acids. acids. Overall, Overall, the theconcentration concentration of organicof organic acids acids in AWP in AWP was was higher higher than than that thatof SWP. of SWP. Form Formicic and andlactic lactic were were the most the mostpredominant predominant organic organic acids acids (299 (299 and and 270 mmol/L,270 mmol respectively)/L, respectively) in AWP. in AWP. This This observation observation is not is notsurprising surprising,, since since acid acid whey whey is derived is derived from fromthe formation the formation of lactic of acid lactic bacteria, acid bacteria, whose main whose product main productis lactic is acid. lactic Aacid. general A general trend w trendas observed was observed in SWP, in SWP,where where the concentration the concentration of organic of organic acids acidsincreased increased slightly slightly with with the reactionthe reaction time time (Figure (Figure 6a).6 a). Citric, Citric, acetic, acetic, and and uric uric acids acids were were found found at atrelatively relatively low low concentrations. concentrations. These These findings findings are are in in agreement agreement with with the earlier studies studies on on the the formationformation of of isosaccharinic isosaccharinic acids acids and organic and organic acids [26 acids]. Organic [26]. Organic acids are acidsformed are from formed the breakdown from the ofbreakdown glucose and of glucose galactose. and Moregalactose. specifically, More specifically, lactic acid lactic is formed acid is as formed the primary as the primary byproduct byproduct of the catalyticof the catalytic oxidation oxidation of glucose of [glucose33]. The [33]. catalytic The c oxidationatalytic oxid of glucoseation of over glucose bimetallic over bimetallic catalysts has catalysts been recognizedhas been recognized as an efficient as an alternative efficient alternative for the production for the production of gluconic of acid.gluconic The acid. oxidation The oxidation of glucose of occurs via a carbonyl conjugated radical mechanism due to the formation of H O , which in turns is glucose occurs via a carbonyl conjugated radical mechanism due to the formation2 2 of H2O2, which in formedturns is by formed the presence by the presence of oxygen of in oxygen the aqueous in the mediaaqueous [34 media]. However, [34]. However, the mechanisms the mechanisms of glucose of oxidationglucose oxidation strongly dependstrongly on depend the pH on of thethe mediumpH of the and medium the type and of catalyststhe type [of33 ].catalysts On the [ other33]. On hand, the theother concentration hand, the concentration of organic acids of organic in AWP acids showed in AWP an increase showed with an timeincrease for citricwith andtime pyruvicfor citric acid, and whilepyruvic the acid, concentration while the concentration of formic and of lactic formic acid and decreased lactic acid with decreased time (Figure with time6b). ( CitricFigure acid 6b). andCitric pyruvicacid and acid pyruvic are formed acid are due formed to glucose due breakdown,to glucose breakdown, which subsequently which subsequently formed oxalic formed acid and oxalic others. acid and others. 3.5. Product Distribution

Figure7 exemplifies the one-pot synthesis of lactose derivatives, where a pool of molecules synthesized from SWP (Figure7a) and AWP (Figure7b) was plotted at di fferent reaction times. In Foods 2020, 9, x FOR PEER REVIEW 9 of 11

3.5. Product Distribution Foods 2020, 9, 784 9 of 11 Figure 7 exemplifies the one-pot synthesis of lactose derivatives, where a pool of molecules synthesized from SWP (Figure 7a) and AWP (Figure 7b) was plotted at different reaction times. In SWP, thethe moremore predominantpredominant compoundscompounds werewere lactose (43–35%),(43–35%), gluconic acid (6 (6–18%),–18%), LAU (14 (14–9%),–9%), formicformic acidacid (8–13%),(8–13%), and lacticlactic acidacid (8–7%),(8–7%), whosewhose concentrationconcentration variedvaried with thethe reactionreaction time.time. For AWP,AWP, lactoselactose (27–23%),(27–23%), lacticlactic acidacid (21–24%),(21–24%), formicformic acidacid (19–20%),(19–20%), gluconicgluconic acidacid (11–7%),(11–7%), andand LAULAU (5–3%)(5–3%) werewere thethe mostmost predominantpredominant compounds.compounds. It is worth mentioning that thesethese resultsresults shouldshould bebe considered cautiouslycautiously becausebecause the finalfinal concentration is influencedinfluenced by a number of factors, including pH,pH, temperature,temperature, the compositioncomposition of thethe stream,stream, thethe typetype ofof catalyst,catalyst, andand catalystcatalyst load.load. Thus,Thus, thethe concentration of of a given a given group group of components of components may be may optimized be optimized in order into maximize order to their maximize production their accordingproduction to according the study ofto reaction the study kinetics. of reaction The concept kinetics. of one-potThe concept was firstof one introduced-pot was by first Kolb introduced et al. [35], whoby Kolb described et al. [35 a], click-chemistry who describedin a click which-chemistry a set of chemical in which transformations a set of chemical yielded transformations higher effi yieldedciency, fasthigher rates, efficiency, and simple fast rates, product and isolation. simple product Soon after,isolation. the conceptSoon after, of clickthe concept chemistry of click quickly chemistry found widespreadquickly found application widespread in various application research in areas,various including research organic areas, chemistry,including organicpolymer che synthesis,mistry, petroleum,polymer synthesis, and biorefinery. petroleum, Currently, and biorefinery. there are severalCurrently, terminologies there are several to describe terminologies multi-step to reactionsdescribe thatmulti take-step place reactions in one that pot, take such place as domino in one pot reaction,, such as cascade domino reaction reaction, and cascade tandem reaction reaction. and One-pot tandem synthesisreaction. One is eff-potective synthesis because is several effective chemical because transformation several chemical steps transformation can be carried steps out incan a singlebe carried pot, whileout in circumventing a single pot, while several circumventing purification procedures several purification at the same procedures time. Thus, at athe one-pot same proceduretime. Thus, can a minimizeone-pot procedure chemical can waste, minimize save time, chemical and simplify waste, save practical time, aspects. and simplify practical aspects.

Figure 7. One-pot synthesis of lactose derivatives from (a) sweet whey permeate and (b) acid Figure 7. One-pot synthesis of lactose derivatives from (a) sweet whey permeate and (b) acid whey whey permeate. permeate. 4. Conclusions 4. Conclusions The one-pot conversion of lactose streams resulted in the formation of four main group of components—rareThe one-pot conversion carbohydrate of lactose (lactulose), streams monosaccharides resulted in the (glucose formation and of galactose), four main sugar group acids of (gluconiccomponents acid),—rare and carbohydrate organic acids. (lactulose), The final distribution monosaccharides and their (glucose concentration and galactose), differ from sugar SWP acids and AWP,(gluconic where acid), lactulose and organic was favored acids. The in SWP, final and distribution organic acids and their were concentration favored in AWP. differ The from synthesis SWP and of a poolAWP, of where molecules lactulose through was a favored one-pot approachin SWP, and represents organic an acids alternative were favored approach in AWP. for the The utilization synthesis of streamsof a pool of lactose. of molecules Upon further through separation, a one-pot organic approach acids represents can be used an as alternative building blocks approach of numerous for the applicationsutilization of instreams the manufacture of lactose. Upon of herbicides, further separation, bioplastics, organic and biofertilizers. acids can be used as building blocks of numerous applications in the manufacture of herbicides, bioplastics, and biofertilizers. Author Contributions: Conceptualization, S.I.M.-M.; methodology, M.E.; analysis, M.E.; writing—original draft preparation,Author Contributions: M.E.; writing—review Conceptualization, and editing, S.I.M. S.I.M.-M.;-M.; methodology, funding acquisition,M.E.; analysis, S.I.M.-M. M.E.; writing All authors—original have draft read andpreparation, agreed to M.E.; the published writing— versionreview and of the editing, manuscript. S.I.M.-M.; funding acquisition, S.I.M.-M. All authors have read Funding:and agreedThis to the work published has been version made of the possible manuscript. through the financial support of the Midwest Dairy Foods Research Center (St. Paul, MN) and partial support from the USDA National Institute for Food and Agriculture (HATCHFunding: projectThis work SD00H607-16). has been made possible through the financial support of the Midwest Dairy Foods Research Center (St. Paul, MN) and partial support from the USDA National Institute for Food and Agriculture (HATCH Conflicts of Interest: The authors declare no conflict of interest. project SD00H607-16).

Foods 2020, 9, 784 10 of 11

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