Article Optimisation of oat milk formulation to obtain fermented derivatives by using probiotic Lactobacillus reuteri microorganisms N Bernat 1 , M Cha ´fer 1 , C Gonza ´lez-Martı ´nez 1 , J Rodrı ´guez-Garcı ´a 2 and A Chiralt 1 Abstract Functional advantages of probiotics combined with interesting composition of oat were considered as an alternative to dairy products. In this study, fermentation of oat milk with Lactobacillus reuteri and Streptococcus thermophilus was analysed to develop a new probiotic product. Central composite design with response surface methodology was used to analyse the effect of different factors (glucose, fructose, inulin and starters) on the probiotic population in the product. Optimised formulation was characterised throughout storage time at 4 C in terms of pH, acidity, b-glucan and oligosaccharides contents, colour and rheological behaviour. All formulations studied were adequate to produce fermented foods and minimum dose of each factor was considered as optimum. The selected formulation allowed starters survival above 10 7 /cfu ml to be considered as a functional food and was maintained during the 28 days controlled. b-glucans remained in the final product with a positive effect on viscosity. Therefore, a new probiotic non-dairy milk was successfully developed in which high probiotic survivals were assured throughout the typical yoghurt-like shelf life. Keywords Fermented oat milk, probiotic, prebiotic, response surface methodology, formula optimisation Date received: 29 July 2013; accepted: 9 December 2013 INTRODUCTION Probiotics are defined as ‘live microorganisms that when administered in adequate amounts confer a health benefit on the host’ (FAO/WHO, 2001). Lactobacillus and Bifidobacterium genus are mostly recognised within this group, although Lactococcus, Enterococcus, Saccharomyces and Propianobacterium genus are currently being investigated (Rivera- Espinoza and Gallardo-Navarro, 2010). However, strains are not classified as probiotic unless they meet some requirements, such as total safety for the host, resistance to gastric acidity and pancreatic secretions, adhesion to epithelial cells, antimicrobial activity, inhibition of adhesion of pathogenic bacteria, stimula- tion of immune system and metabolic activity, evalu- ation of resistance to antibiotics, tolerance to food additives, technological procedures or stability in the food matrix (Prado et al., 2008). The use of bacteria in food product manufacturing dates back to the ancient world, although the purposes have changed. Nowadays, probiotic microorganisms are used in order to develop fermented products which are able to exert nutritional and healthy benefits, 1 Institute of Food Engineering for the Development, Universitat Polite `cnica de Vale `ncia, Valencia, Spain 2 Research Group of Food Microstructure and Chemistry, Universitat Polite `cnica de Vale `ncia, Valencia, Spain Corresponding author: N Bernat, Institute of Food Engineering for the Development, Universitat Polite `cnica de Vale `ncia. Edificio 8E. Planta 3, Camino de vera s/n, 46022 Valencia, Spain. Email: [email protected]Food Science and Technology International 21(2) 145–157 ! The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav DOI: 10.1177/1082013213518936 fst.sagepub.com by guest on April 5, 2016 fst.sagepub.com Downloaded from
Transcript
Article
Optimisation of oat milk formulation to obtain fermentedderivatives by using probiotic Lactobacillus reuterimicroorganisms
N Bernat1, M Chafer1, C Gonzalez-Martınez1,J Rodrıguez-Garcıa2 and A Chiralt1
AbstractFunctional advantages of probiotics combined with interesting composition of oat were considered as analternative to dairy products. In this study, fermentation of oat milk with Lactobacillus reuteri andStreptococcus thermophilus was analysed to develop a new probiotic product. Central composite designwith response surface methodology was used to analyse the effect of different factors (glucose, fructose,inulin and starters) on the probiotic population in the product. Optimised formulation was characterisedthroughout storage time at 4 �C in terms of pH, acidity, b-glucan and oligosaccharides contents, colourand rheological behaviour. All formulations studied were adequate to produce fermented foods and minimumdose of each factor was considered as optimum. The selected formulation allowed starters survival above107/cfu ml to be considered as a functional food and was maintained during the 28 days controlled. b-glucansremained in the final product with a positive effect on viscosity. Therefore, a new probiotic non-dairy milk wassuccessfully developed in which high probiotic survivals were assured throughout the typical yoghurt-likeshelf life.
KeywordsFermented oat milk, probiotic, prebiotic, response surface methodology, formula optimisation
Date received: 29 July 2013; accepted: 9 December 2013
INTRODUCTION
Probiotics are defined as ‘live microorganisms thatwhen administered in adequate amounts confer ahealth benefit on the host’ (FAO/WHO, 2001).Lactobacillus and Bifidobacterium genus are mostlyrecognised within this group, although Lactococcus,Enterococcus, Saccharomyces and Propianobacteriumgenus are currently being investigated (Rivera-Espinoza and Gallardo-Navarro, 2010). However,strains are not classified as probiotic unless they meetsome requirements, such as total safety for the host,resistance to gastric acidity and pancreatic secretions,adhesion to epithelial cells, antimicrobial activity,
inhibition of adhesion of pathogenic bacteria, stimula-tion of immune system and metabolic activity, evalu-ation of resistance to antibiotics, tolerance to foodadditives, technological procedures or stability in thefood matrix (Prado et al., 2008).
The use of bacteria in food product manufacturingdates back to the ancient world, although the purposeshave changed. Nowadays, probiotic microorganismsare used in order to develop fermented productswhich are able to exert nutritional and healthy benefits,
1Institute of Food Engineering for the Development, UniversitatPolitecnica de Valencia, Valencia, Spain2Research Group of Food Microstructure and Chemistry,Universitat Politecnica de Valencia, Valencia, Spain
Corresponding author:N Bernat, Institute of Food Engineering for the Development,Universitat Politecnica de Valencia. Edificio 8E. Planta 3, Caminode vera s/n, 46022 Valencia, Spain.Email: [email protected]
Food Science and Technology International 21(2) 145–157! The Author(s) 2014 Reprints and permissions:sagepub.co.uk/journalsPermissions.navDOI: 10.1177/1082013213518936fst.sagepub.com
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such as reduction of hypercholesterolemia, hostimmune modulation, prevention of urogenital diseases,alleviation of constipation, protection against travel-ler’s diarrhoea, protection against colon and bladdercancer or prevention of osteoporosis and food allergies(Lourens-Hattingh and Viljoen, 2001). Nevertheless,host benefits are subject to the strain type used in prod-uct manufacture (Sharareh et al., 2009). Although thereis no legal definition of the term ‘probiotic’, differentprobiotic’s dosage recommendations can be found.According to different authors, the minimum numberof viable probiotic bacteria should be 107–109 colonyforming units (cfu)/g or ml of a product at the time ofconsumption and, as to exert healthy effects, it shouldbe consumed daily (Gomes and Malcata, 1999; Stantonet al., 2003; Van Niel et al., 2002). These recommenda-tions are in compliance with the minimum requirementsfor standard milk fermented products by theInternational Dairy Federation and the Japan andEU Associations of Fermented Milks, which is 107
cfu/g or ml of starter (Sanz and Dalmau, 2008).Due to their health properties, there has been a not-
able increase in the consumption of food products con-taining probiotic microorganisms. As Granato et al.(2010) reported, the consumption of probiotic productsincreased by around 13 and 18% between 2002 and2007 in Eastern and Western Europe, respectively.Those products have been traditionally produced byusing animal milk, yoghurt being the best known.Nonetheless, new food matrices have been investigated,such as meat, baby food, ice creams, juices and cereals(Granato et al., 2010) to produce probiotic products. Inthis sense, several beverages obtained from soy, rice,wheat and maize would have huge market potentialdue to the current consumer demand for cow-milk sub-stitute products (Martensson et al., 2000). Those con-sumers are principally vegetarians, people allergic toanimal proteins or lactose intolerant. In this sense,oat milks can be alternative matrices with which toelaborate probiotic products, adding the nutritionaland functional characteristics of this cereal, such asthe high content in soluble and non-soluble fibrewhich makes oats a useful product to use in the pre-vention of different diseases, especially those affectingthe colon (Sadiq-Butt et al., 2008). Several studies haveshown that b-glucans, the most prevalent oat solublefibre, have prebiotic activity, while they decrease theblood cholesterol levels, cardiovascular disorders andimprove the lipid and glucose metabolism. Prebioticsare non-digestible components of functional foodsthat stimulate the proliferation and activity of bacterialpopulations desirable in the colon and inhibit pathogenmultiplication; hence acting beneficially on the host(Mattila-Sandholm et al., 2002; Roberfroid, 2004).b-glucans are seen to be able to stimulate the growth
of the health-promoting intestinal microflora, with aparticular effect on lactic acid bacteria andBifidobacteria genus (Angelov et al., 2006).
Regardless of the health benefits of oat consump-tion, its sensorial properties lead to low consumeracceptance. Nevertheless, both technological and fer-mentative processes are bound to improve sensorialquality, as well as providing health and nutritionalbenefits due to the combination of both probiotic andprebiotic compounds, the so-called synbiotics.Lactobacillus reuteri ATCC 55730 is a well-establishedprobiotic strain (Casas and Mollstam, 1997) whichcould be used to develop synbiotic products. Hence,these kind of products could be beneficial for targetedgroups such as the paediatric population, since this pro-biotic bacteria has been reported to improve symptomsof infantile colic (Savino et al., 2007), feeding toleranceand gut functions in pre-term infants (Indrio et al.,2008), reduced constipation (Coccorullo et al., 2010)and modulate cytokine patterns involved in atopic dis-eases (Miniello et al., 2010). L. reuteri ATCC 55730 hasbeen already used to develop fermented oat-based bev-erages (commercially called Adavena) by Martenssonet al. (2002). In the study, these authors used this pro-biotic L. reuteri (and other probiotic microorganismssuch as Bifidobacterium bifidum DSM 20456 andLactobacillus acidophilus DSM 20079) as pure starteror mixed with the typical yoghurt bacteria(Streptococcus thermophilus and Lactobacillus del-brueckii subsp. bulgaricus) to develop the fermentedproducts, where survival counts and changes in glucose,maltose and b-glucan contents throughout storage timewere quantified. The results showed that these oat-based milks can be used to support the growth of thebacteria, which maintained high cell viability duringcold storage; the highest viability was found whenusing L. reuteri. The b-glucan content remained unaf-fected for the L. reuteri, L. acidophilus and for the yog-hurt culture through fermentation process or storage,thus contributing to maintain its functionality as pre-biotic in the developed products. Therefore, consider-ing these results, L. reuteri can be used to developsuitable new yoghurt-type oat products with synbioticfeatures. Moreover, taking into account the stimulatingeffect of S. thermophilus on the growth of L. delbrueckiiin standard yoghurts (Tamime and Robinson, 2002),the combination of L. reuteri with S. thermophilusmight improve the fermentation process of oat milks.
The aim of the present study was to evaluate thefermentative process of oat milks (Avena sativa L.)with the mixed culture L. reuteri ATCC 55730 andS. thermophilus CECT 986 (1:1) and the quality of thefermented product. To this end, the effect of differentfactors, such as the added amount of glucose, fructose,inulin and inoculum, was analysed to ensure there was
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enough viable probiotic strain (L. reuteri) in the finalproduct. The most adequate fermented formulationwas characterised in terms of their main physicochem-ical properties in order to determine the product’s qual-ity throughout storage time.
MATERIALS AND METHODS
Preparation of oat milk
Oat milk was produced by soaking and grinding peeledoat (A. sativa L.), supplied by Salud e Imaginacion S.L.(Masquefa, Barcelona, Spain). The oat:water ratio was8:100 (w/v), which ensures enough quantity of b-glucan(oat prebiotic compound) for the subsequent fermenta-tive process (Angelov et al., 2006). The extraction wascarried out in Starsoja (Farmanutrients Labs, S.L.,Barcelona, Spain), equipment specifically designed forthe production of vegetable milks. To obtain the oatmilk, three grinding cycles were used at 90 �C for20min. The liquid obtained was then homogenised ina rotor–stator homogeniser (Ultraturrax T25, Jankeand Kunkel, Germany) for 3min at 13,500 r/min and,finally, sterilised at 121 �C for 15min (Presoclave II, JP-Selecta, Barcelona, Spain).
Preparation of fermented products
Inoculum preparation. L. reuteri ATCC 55730 and S.thermophilusCECT 986 were activated from their frozenforms (stored in 40 g/100ml glycerol at �80 �C), bytransferring each one to its selective broth until optimalbacterial growth is assured. Selective broths were Man,Rogosa and Sharpe (MRS) (Scharlab, Barcelona,Spain) for the probiotic Lactobacillus and M17(DifcoTM, New Jersey, USA) for S. thermophilus.Incubation conditions were 37 �C/24 h/anaerobicallyfor L. reuteri, in which anaerobiosis was created byusing anaerobic jars and a CO2-generator system(AnaeroGenTM, Oxoid Ltd, Basingstoke, England),and 42 �C/24 h/aerobically for S. thermophilus.
Likewise, strains were independently incubated intheir broths for 24 h and then centrifuged at 8600g�10min at 4 �C; supernatant was discarded.Immediately after, bacteria were resuspended in PBS1� buffer (10mM phosphate, 137mM NaCl, 2.7mMKCl, pH 7.4) until they reached concentrations of108 cfu/ml.
Experimental design for fermentation process.Amount of glucose, fructose, inulin and starter inocu-lum were selected as factors (four independent vari-ables) to obtain fermented oat milks. Centralcomposite design (CCD) with randomised response sur-face methodology (RSM) was used to analyse the effectof the different factor combinations on the total count
of probiotic bacteria (response variable) and then opti-mise fermentation process, such as described by otherauthors (Chen et al., 2004; Cruz et al., 2010; Guptaet al., 2010; Liew et al., 2005; Stepheine et al., 2007;Yaakob et al., 2012). Statistical analysis of the datawas carried out in Statgraphics� Centurion XVI byusing an orthogonal 24þ star design, which studiedthe effects of the four factors in 31 runs. Factors wereselected considering several aspects: glucose is a properand cheap carbon source for the growth of probiotic L.reuteri, fructose addition allows both, to prevent theheterofermentative pathway of L. reuteri which endsin ethanol synthesis and to improve the strain growthdue to the role of this sugar as e� acceptor that enablesto a better balanced redox system (Arskold et al., 2008);inulin was selected due to its reported health benefits(Kolida et al., 2002) that would raise the added value ofthe oat fermented product focus of the study.Furthermore, it might stimulate the probiotic survivalduring cold storage, as it has been stated by otherauthors (Desai et al., 2004; De Souza-Oliveira et al.,2009). The levels of the factors were chosen takinginto account previous studies into oat fermentation stu-dies (Angelov et al., 2006; Sumangala et al., 2005):Glucose: 1–2 g/100ml, fructose: 1–2 g/100ml, inulin:0.7–1.3 g/100ml and inoculum: 3–4.5ml/100ml. Theresponse variable was the probiotic population at theend of fermentation process.
Fermentation process in the 31 runs was carried outby adding the corresponding amount of starter culture(prepared by mixing in a 1:1 volume ratio the L. reuteriand S. thermophilus PBS buffer suspensions) to the for-mulated and sterilised oat milks and then incubating at40 �C, which was the optimal growth temperature of themixed culture, according to a preliminary study (datanot shown). Fermentation process was stopped whenpH of samples reached 4.4–4.6, by cooling the samplesat 4 �C, which was the storage temperature until theanalyses were done.
A step-wise second grade polynomial fitting wasused to model the response variable as a function ofthe factors.
Optimal formulation of the fermented product wasestablished on the basis of the obtained results for theresponse variable.
Product characterisation
Newly obtained oat milk and optimal formulation offermented product stored at different times were char-acterised as to content in different sugars and b-glucan(prebiotic), pH, acidity, density, colour, rheologicalbehaviour and microstructure. In oat milk, drymatter, protein, lipid and ash contents were also ana-lysed. In fermented product, the starter survival
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throughout storage time (1, 7, 14, 21 and 28 days) at4 �C was analysed. All the analyses were done intriplicate.
Chemical analyses. Association of Official AnalyticalChemists (AOAC) official methods of analysis wereused to determine moisture (AOAC 16.006), total nitro-gen (AOAC 958.48) and fat contents (AOAC 945.16)(Horwitz, 2000). Ashes were obtained following theprotocol reported by Matissek et al. (1998).
Total b-glucan content was determined enzymaticallywith a mixed-linkage b-glucan detection assay kit(Megazyme TM International Ltd, Wicklow, Ireland).
Sugar profiles were analysed and the different sugarswere quantified using the following High-PerformanceAnion-exchange Chromatography with PulsedAmperometric Detection (HPAC-PAD) equipment:Metrohm 838 Advanced Sample Processor(Metrohm� Ltd, Herisau, Switzerland) in anAdvanced Compact IC 861 ion chromatograph (IC)equipped with a pulsed amperometric detector to moni-tor the separation (Bioscan 817). Prior to the analysis,samples were diluted 1:100 with nanopure water.Sample proteins were removed by precipitation withglacial acetic acid and centrifugation at 10,000 r/minfor 10min; pH was then reconstituted at initialvalues. Before injecting samples into the equipment,they were filtered through nylon membranes(0.45 mm). A Metrosep CARB guard column(5mm� 4.0mm Metrohm) and a Metrosep CARB 1(250mm� 4.6mm Metrohm) analyses column wereused. A total of 20 ml of sample were injected andeluted (1ml/min) with NaOH 0.1M, at 32 �C. An Auworking electrode was used and applied potentials wereþ0.05V (between 0 and 0.40 s), þ0.75V (between 0.41and 0.60 s) and þ0.15V (between 0.61 and 1 s).Software ICNet 2.3 (Metrohm� Ltd) was used fordata collection and processing. The concentration ofeach sugar was determined from their respective cali-bration curves, obtained from standard solutions ofmannitol, glucose, fructose and sucrose (Sigma-Aldrich�, Spain), done in triplicate.
pH, density (�) and titratable acidity (TA).Measurements of pH and r were carried out at 25 �Cusing a pH meter (GLP 21þ, Crison Instruments S.A.,Barcelona, Spain) and a picnometer Gay-Lussac,respectively. AOAC standard method was used todetermine TA of samples (AOAC 947.05), expressingresults as g/100ml of lactic acid (Horwitz, 2000).
Rheological behaviour. The rheological behaviour wascharacterised in a rotational rheometer (HAAKERheostress 1, Thermo Electric Corporation,Germany) with a sensor system of coaxial cylinders
type Z34DIN Ti. The shear stress (s) was measuredas a function of shear rate ( _�) from 0 to 112 s�1,using 1min to reach the maximum shear rate andanother minute to attain zero shear rate. Non-linearmodel (equation (1)) was applied to determine theflow behaviour index (n), consistency index (K) andyield stress (sy). Apparent viscosities were calculatedat 50 s�1 (equation (2)), since shear rates generated inmouth when food is being chewed and swallowed arebetween 10 and 100 s�1 (McClements, 2004)
� ¼ �y þ K _�n ð1Þ
� ¼ K � _�n�1 ð2Þ
Colour parameters. Colour coordinates were mea-sured from the reflection spectrum in a Spectrum-col-orimeter CM-3600 d (MINOLTA Co., Osaka, Japan).A 20-mm depth cell was used. CIE L*a*b coordinateswere obtained using illuminant D65/10� observer.Lightness (L*), chrome (C�ab) and hue (h�ab) of the dif-ferent samples as well as colour difference (�E) (equa-tions (3) to (5)) with respect to the non-fermentedsample were obtained
Confocal laser scanning microscopy. A Nikon con-focal microscope C1 unit, which was fitted on aNikon Eclipse E800 microscope (Nikon, Tokyo,Japan), was used. An Ar laser line (488 nm) wasemployed as light source to excite fluorescent dyesRhodamine B and Nile Red. Rhodamine B (Fluka,Sigma-Aldrich, Missouri, USA) with �ex max 488 nmand �em max 580 nm was dissolved in distilled water at0.2 g/100ml. This dye was used to stain proteins andcarbohydrates. Nile Red (Fluka, Sigma-Aldrich,Missouri, USA) with �ex max 488 nm and �em max515 nm was dissolved in Polyethylene glycol (PEG)200 at 0.1 g/l. This dye was used to stain fat. An oilimmersion objective lens (60�/1.40NumericalAperture (NA)/Oil/Plan Apo VC Nikon) was used.
For sample visualisation a microscopy slide was ela-borated with two razor blades (platinum-coateddouble-edge blades with 0.1mm thickness) stuck to aglass. A total of 20 ml of the sample were placed on themicroscope slide, within the central gap of the blades;10 ml of Rhodamine B solution and 10 ml of Nile Redsolution were added and the cover slide was carefully
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positioned. Observations were performed for 10minafter diffusion of the dyes into the sample. Imageswere observed and stored with 1024� 1024 pixel reso-lution, using the microscope software (EZ-C1 v.3.40,Nikon, Tokyo, Japan).
Starter survival. Counts of L. reuteri and S. thermophi-lus were performed using pour plate technique, accord-ing to the method described by the International DairyFederation (International IDF Standard, 1997). M17agar (DifcoTM, New Jersey, USA) selective media wasused for S. thermophilus and MRS agar (Scharlab,Barcelona, Spain) for L. reuteri, which was acidifiedto pH 5.4 with acetic acid to prevent possible growthsof S. thermophilus. Incubation conditions were 37 �Cfor 48 h in aerobic conditions for S. thermophilus and37 �C for 24 h in anaerobic conditions for L. reuteri;anaerobiosis was created by using anaerobic jars anda CO2-generator system (AnaeroGenTM, Oxoid Ltd,Basingstoke, England).
Statistical analysis
Results were analysed by multifactor analysis of vari-ance with 95% significance level using Statgraphics�
Centurion XVI. Multiple comparisons were performedthrough 95% Fisher’s Least Significant Differenceintervals (LSD) intervals.
RESULTS AND DISCUSSION
Characterisation of the oat milk: Chemicalcomposition and microstructure
Results in oat milk chemical composition were:6.5� 0.3 g/100ml of dry matter, 0.65� 0.03 g/100mlof proteins, 0.241� 0.004 g/100ml of b-glucan,0.094� 0.003 g/100ml of fats, 0.099� 0.005 g/100mlof ashes and 0.047� 0.007 g/100ml of total sugars,the latter obtained from the sum of all the individualsugars analysed. These compositional values are inagreement with those reported by other authors(Sadiq-Butt et al., 2008). As was observed during theextraction process, the major losses of oat componentsoccur in the fibre and lipid fractions, which remained inthe waste by-product during the extraction process.This fact is coherent with that observed in the oatmilk microstructure, by using the confocal technique(Figure 1(a) and (b)), where the presence of a smallamount of lipid droplets, yellow-green in colour, canbe observed. Proteins and carbohydrates appear red-coloured, together with some cellular fragments. Asconfocal pictures show, the oat milk’s microstructureis organised as a polysaccharide network (PM) wherefat and protein are embedded. This arrangement isassociated to the gelling properties of b-glucans, once
it is heated (Lazaridou and Biliaderis, 2007. Moreover,almost all the lipid droplets are retained in the polysac-charide–protein matrix which is responsible for thephysical stability of the oat milk, even after the steril-isation treatment. It was observed that some proteinswere attached to fat globules, thus providing protectionagainst the so-called Ostwald ripening or other desta-bilisation processes in emulsions.
Effect of factors on fermentation process
Table 1 presents the experimental values of the L. reu-teri counts (log cfu/ml) obtained for each run of theCCD. All the formulations permitted the developmentof probiotic oat fermented milk, since their responsevariables were over 7 log cfu/ml, which is within theprobiotic amount recommended to ensure health effectsin consumers (Gomes and Malcata, 1999; Stantonet al., 2003; Van Niel et al., 2002).
Results from the 31 runs were fitted to a second-order polynomial equation and the removal of non-sig-nificant terms (P> 0.05) was applied, except when theelimination of such terms decreased the explained vari-ance (R2 adj). The goodness of the fitted model wasevaluated through an analysis of variance, mainlybased on the F-test and on the R2adj, which providea measurement of how much of the variability in theobserved response values could be explained by theexperimental factors and their interactions (Granatoet al., 2010). Table 2 summarises the estimated regres-sion coefficients of the second-order model obtained, inwhich fit parameters from the analysis of variance areincluded.
As can be seen in the coefficients (Table 2), whenthe factors appear as linear variables, they seem tonegatively affect the total probiotic counts.Nevertheless, a more thorough examination of thefitted model indicated that all the coefficients corres-ponding to factor interactions (second order terms)were positive in value, which explains the overall posi-tive impact of the increasing levels of glucose, fruc-tose, inulin and starter inoculums on the totalprobiotic counts. These results indicated that the indi-vidual factors were not truly independent of oneanother, which is statistically known as ‘multicoli-nearity’ and represents a common problem in regres-sion analyses (Bender et al., 1989). Whenmulticolinearity occurs, the elimination of non-significant explanatory variables in the model is notrecommended (Bender et al., 1989).
As regards the model fit, the lack-of-fit parameterwas not significant (P> 0.05) and the P value of theDurbin–Watson statistic was greater than 0.05, mean-ing that there is no indication of serial autocorrelationin the residuals at the 5% significance level.
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Both parameters indicated that the obtained model isadequate for predicting probiotic survival in oat milk.In practice, a model is considered adequate to describethe influence of the variable(s) when the coefficient ofdetermination (R2) is at least 80% (Yaakob et al., 2012)or the values of R2adj over 70% (Cruz et al., 2010). Theobtained model explained only 51% of the variation inthe experimental data (R2adj) (Table 2), which is par-tially explained by the narrow range of experimentalresponse variable (&2.5 log cfu/ml). The narrow vari-ation in response variable made it difficult to obtaingreater R2 adj values. Therefore, the obtained predic-tion model should only be used to make roughpredictions.
Figure 2 shows the response surface plots for the L.reuteri counts. Four different plots were obtained ineach of which one of the factors was fixed at the smal-lest value. As deduced from Table 1, surfaces showedthat many formulations could be used for the produc-tion of probiotic fermented oat milk, by considering theminimum recommended strain survival (�107 cfu/ml).Taking these results into account, a possible optimumformulation should be defined as one that has the
minimum production costs. In this sense, the formula-tion considered as optimum was the one to whichthe smallest amount of ingredients was added. Thisoptimum corresponds to the formulation where0.65 g/100ml of glucose, 0.65 g/100ml of fructose,0.4 g/100ml of inulin and 3ml/100ml of mixed cultureinoculum were incorporated into the oat milk.
This optimal formulation was submitted to fermen-tation process and data were analysed in order to val-idate the prediction model. Results showed that thefermented product reached 4.37� 0.02 value of pH in3.5 h at 40 �C with a L. reuteri survival of around 9 logcfu/ml. Martensson et al. (2002) reported longer fer-mentation times (16 h) in commercial oat beverages,additionally enriched or not with glucose (0, 4.4 and8.8 g of glucose/100 g beverage), when L. reuteri wasused as pure or mixed starter, probably due to themuch lower yoghurt bacteria concentration used(0.02%, ratio L. delbrueckii subsp. Bulgaricus: S. ther-mophilus 1:1). Moreover, lower probiotic survivals werereported, around 8 log cfu/ml, when it was used as purestarter and one log less, when it was combined with thetypical yoghurt starter bacteria.
Figure 1. Confocal pictures of both sterilised (a and b) and fermented oat milk (c and d). CP: continuous phase; f: fatdroplet; p: protein; PM: polysaccharide–protein matrix; S: starter bacteria.
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Bacterial counts and acid production. Average valuesof pH and TA in fermented oat milk versus storage timeare summarised in Table 3. This table also includesbacterial count values of L. reuteri and S. thermophilus(log cfu/ml) throughout storage time.
As can be seen from Table 3, physicochemicalproperties of oat milk were modified due to
fermentation process. pH of the fermented productremained within the desired ranges (above 4) for thefirst day but, after 7 days, pH significantly decreasedreaching a unit below the initial value. These changeswere expected considering that starters were viable overthe entire storage time and, therefore, they were stillgenerating acidic compounds. The same trend wasobserved in TA assays, since the values quantified inoat fermented products increased throughout the stor-age time (Table 3). Initial acidity of the oat fermentedmilk averaged 0.17 g/100ml of lactic acid and increasedthroughout cold storage time, reaching values up to0.5 g/100ml in the last time controlled. Compared tocow milk fermentations, TA values were lower thanin standard yoghurt, in which it is around 0.8–1 g oflactic acid/100 g (Mistry and Hassan, 1992; Tamimeand Robinson, 2000) due to the greater buffering cap-acity of cow milk.
With regards to the bacteria survivals, although theviability of starter bacteria decreased during the storagetime, especially in L. reuteri, the minimum survival rec-ommended, 107 cfu/ml (Sanz and Dalmau, 2008), wasensured in both strains until the end of the storage.Results show that both microorganisms are highlyresistant to an acidic environment and that the oat for-mulation contained sufficient nutrients for startergrowth during the whole storage time. Previous workshave also shown good fermentation results of L. reuteri
Table 1. Total counts of L. reuteri obtained in the differentfermented products corresponding to the experimentaldesign, as a function of the levels of the factors
FactorsResponsevariable
Run order X1 X2 X3 X4 Y
1 0 þa 0 0 8.04
2 0 0 þa 0 8.23
3 �1 þ1 �1 þ1 8.04
4 0 0 0 0 8.26
5 þ1 þ1 �1 þ1 9.00
6 0 0 0 0 8.08
7 0 0 0 0 8.36
8 �a 0 0 0 9.15
9 �1 þ1 þ1 þ1 8.73
10 �1 þ1 �1 �1 9.20
11 þ1 �1 �1 þ1 8.95
12 þ1 �1 þ1 �1 7.46
13 0 0 0 0 9.34
14 þa 0 0 0 10.25
15 �1 �1 þ1 �1 8.81
16 þ1 þ1 þ1 �1 9.60
17 þ1 �1 �1 �1 8.76
18 0 0 0 0 9.20
19 0 0 �a 0 9.34
20 þ1 þ1 þ1 þ1 9.28
21 0 0 0 þa 8.67
22 �1 þ1 þ1 �1 9.11
23 0 �a 0 0 9.46
24 �1 �1 �1 þ1 9.28
25 þ1 �1 þ1 þ1 8.21
26 0 0 0 0 8.08
27 0 0 0 0 8.26
28 þ1 þ1 �1 �1 8.32
29 0 0 0 �a 8.54
30 �1 �1 �1 �1 9.83
31 �1 �1 þ1 þ1 8.15
Factors X1, X2, X3, X4 and Y stand for glucose: 10–20 g/l; fructose:10–20 g/l; inulin: 7–13 g/l; inoculum: 30–45 ml/l; probiotic counts(log cf/ml), respectively.
Table 2. Regression coefficients and analysis of variancefor probiotic counts (log cfu/ml) obtained from the fittedmodel
Factor/parameterRegressioncoefficient/value
Constant 22.18
Glucose �8.22
Fructose �3.81
Inulin �4.11
Inoculum �1.14
Glucose�glucose 1.25
Glucose� fructose 0.95
Glucose� inoculum 0.76
Fructose� inulin 2.38
P value lack of fit 0.880
R2 0.59
R2-adj 0.51
Standard error of est. 0.53
Mean absolute error 0.32
Durbin–Watson statistic (P value) 1.678 (0.218)
R2¼ coefficient of determination; R2-adj¼ explained variance.
Bernat et al.
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by using oat-based formulas as culture media(Johansson et al., 1993; Martensson et al., 2002).
Sugar and �-glucan contents. The concentrationvalues of these components can provide interestinginformation about bacterial activity during the productshelf life. Table 4 presents the values of each sugaridentified in oat fermented product and their changesthroughout the storage time.
Prior to the fermentation process, natural sucrose(from oat), glucose and fructose (added) were presentin oat milk. Other oligosaccharides observed in chro-matograms, which could not be identified, could comefrom the added inulin, taking into account the thermalstability of b-glucans (Lazaridou and Biliaderis, 2007).They were classified as fructans, which is a term thatincludes both inulin and its derivatives (Roberfroid,2004). After the fermentation process, a huge reduction
Figure 2. Response surface plots of the effect of the different factors (glucose, fructose, inulin and starters inoculum) onthe viability of L. reuteri (expressed in log cfu/ml). Plots were obtained by keeping the level of one factor constant. (a)Estimated response surface mesh, glucose¼ 0.65; (b) estimated response surface mesh, fructose¼ 0.65; (c) estimatedresponse surface mesh, inulin¼ 0.4; (d) estimated response surface mesh, inoculum¼ 3.0.
Table 3. Values (mean (SD)) of pH, TA and bacterial counts of fermented oat milk throughout storage time at 4 �C. Dataof non-fermented oat milk are included for comparisons
in the contents of initial monosaccharides and sucrosewas observed and subsequently, a significant (P< 0.05)decrease, gradual throughout storage time, wasobserved (Table 4). These results are expected, sincebacteria starters were viable during the whole storageperiod and they consumed sugars as nutrients (Table 3).On the other hand, a new peak appeared in fermentedproducts that was not present in non-fermented milk,which was identified as mannitol. The presence of man-nitol could be an added value to the oat fermentedproduct developed, since it is seen to have antioxidantproperties (Wisselink et al., 2002).
The appearance of mannitol is attributed to the het-erofermentarive metabolic pathway of the L. reuteristrain used, in which fructose is used as an e� acceptorto regenerate Nicotineamide-Adenine-Dinucleotide(NAD) resulting in mannitol as the end product(Arskold et al., 2008; Von Weymarn et al., 2002).Mannitol yields (mol mannitol produced per mol fruc-tose consumed) quantified are presented in Table 4. Ascan be observed, initially the oat fermented producthad a mannitol yield of almost 90%, in agreementwith other reported values (Von Weymarn et al.,2002). Nevertheless, this yield decreased throughoutthe storage time. The limited glucose content in thematrix from the 7th day of storage (Table 4) mighthave caused also the use of fructose as a C-source(instead of as an e� acceptor), thus decreasing/stoppingthe mannitol production.
As has been mentioned, glucose was a selective nutri-ent for starter bacteria, since it was the one whichdecreased most after fermentation. Indeed, this sugarwas not present in 14-day fermented products.Surprisingly, fructose did not have the same tendencyas glucose, which could be explained by consideringthat starters might have hydrolysed inulin (unions offructose) for nutrition purposes. Inulin and its deriva-tives are seen to be able to stimulate the growth and/ormetabolic activity of bacteria, mainly the genus of
bifidobacteria and lactobacilli (Gibson et al., 2004).This assumption is reinforced by the qualitative ana-lysis of the obtained chromatograms, since the area offructans’ peaks decreased at the end of the storage(data are not shown).
Regarding b-glucan content (data not shown infigure), oat milk initially contained 0.241� 0.004 g/100 g.Once the fermentation process ended, the initial con-centration significantly (P< 0.05) decreased &17%,despite the thermal and acidic stability of b-glucans(Lazaridou and Biliaderis, 2007; Velasco et al., 2009).Therefore, starter bacteria might have hydrolysed thiscompound in order to obtain nutrients for their growth.Nevertheless, b-glucan content in fermented productsdid not change significantly throughout storage time,reaching an average value of 0.199� 0.008 g/100 g.Results reflected that the starter bacteria did not havepreferences in this polysaccharide and, from the devel-opment of the concentration of analysed sugars, theycould also use the added inulin for their survival.Nevertheless, this fact is positive for the final product,since b-glucan prebiotic properties are still available inthe product and are beneficial for consumer health.
Microstructure. Figure 1(c) and (d) shows pictures offermented oat milk microstructure obtained in confocalmicroscope. The main difference in initial oat milkmicrostructure (Figure 1(a) and (b)) was the presenceof a cloudy red area (S) that could be the starter bac-teria. Moreover, much smaller amounts of proteinswere observed; the starters might have hydrolysedthem so as to obtain amino acids for their nutrition.Nonetheless, and in spite of having observed some coa-lesced fat droplets, the major fat component was stillintegrated in the structure in PM network, which waspositive for the product’s physical stability.
Physical properties. No statistical differences werefound between the density parameters (r) of fermented
Table 4. Concentrations of the different sugars identified in fermented oat milk and mannitol yield (mean values and(SD)) throughout storage time. Concentrations of sugars identified in non-fermented oat milk are included forcomparisons
samples after different storage times, the mean valuebeing 1043� 3 kg/m3. Nevertheless, fermentationcauses a slight increase in r, since values for non-fermented milk were 1019� 9 kg/m3. Starters couldmodify the inner structure of oat milks, probably dueto their proteolytic activity, inferred both from the ana-lysis of their microstructure (Figure 1) and from pos-sible changes in charge within the product matrix.
Rheological parameters play a key role in the defin-ition of textural and sensorial perception of a new prod-uct. These parameters were obtained by using a non-linear regression procedure to fit equation (1) to theflow curves of fermented and non-fermented oat milksand are summarised in Table 5. The apparent viscosityof samples at 50 s�1 shear rate was also shown.
Both fermented and non-fermented oat milks wereclassified as plastic, since samples showed yield stressand flow behaviour index (n) values <1. Other authorsalso observed shear thinning behaviour in cerealb-glucan aqueous dispersions (Lazaridou andBiliaderis, 2009; Vasiljevic et al., 2007; Velasco et al.,2009). The fermentation process modified the originalrheological behaviour of oat milk (P< 0.05), increasingthe apparent viscosity of the samples. This parameterdid not show significant changes throughout the stor-age time (P< 0.05). Although proteins seem to behydrolysed by the starter bacteria (Figure 1) and thefermented samples had a lower b-glucan concentrationthan the non-fermented (&17% less), the remaining oatb-glucans showed a thickening and gelling capacity,which means they have the ability to increase the
viscosity of aqueous solutions. Several authors(Lizaridou and Biliaderis, 2009) reported that thelower the molecular weight of the b-glucan, the greateris its gelling capacity, which can be attributed to thehigher mobility of the shorter chains that enhances dif-fusion and lateral interchain associations. Indeed,Piotrowska et al. (2009) and Sahan et al. (2008)observed that b-glucan additions to yoghurt productionimproved sensory properties and physical stability atconcentrations of 0.3 and 0.5 g/100ml. Besides theeffect of b-glucans on the final viscosities of fermentedproducts, L. reuteri is able to synthesise exopolysac-charides which might contribute to the observedincrease in viscosity (Arskold et al., 2007).
Although storage time was not observed to have asignificant effect on rheological parameters, a little syn-eresis was observed after 28 storage days, coherent withthe progressive aggregation of the particles forming thedispersed phase.
The colour parameters of the samples are presentedin Table 6, in which the mean values and standard devi-ation of colour coordinates of fermented and non-fer-mented samples are shown. The different values infermented samples were not observed to differ signifi-cantly (P< 0.05) throughout the storage time, and sothe average values of all fermented samples wereincluded in the table.
The structural changes caused by fermentation werereflected in the optical properties of fermented oat milk,since significant differences were observed in colourparameters between non-fermented and fermented
Table 5. Mean values and standard deviation of consistency index (K), flow behaviour index (n) and yield stress (sy) offermented oat milk throughout storage time obtained from fitting experimental data to non-linear model (non-linear cor-relation coefficient R2 is included). Apparent viscosity (Z) was calculated at shear rate of 50 s�1. Data of oat milk areincluded for comparisons
Storage time (days) K (Pa�sn) n sy Pa) R2 Z50 Pa�s)
a, b, c, d: Different letters in same column indicate significant differences between samples analysed (95% of confidence).
Table 6. Mean values and (standard deviation) of lightness (L*), colour coordinates a* and b*, hue (h�ab), chrome (C�ab)and colour difference (�E) between non-fermented and fermented oat milks
products. Lightness and hue parameters increased afterthe fermentation process, while chrome decreased(P< 0.05). Nevertheless, the total colour differencebetween fermented and non-fermented oat milks (�E)was very small since, according to Francis (1983),values lower than 3 units cannot be easily detected bythe human eye.
CONCLUSIONS
The RSM was used to identify the levels of the differentfactors which permit an optimal oat milk formulationfor the fermentation process to be obtained with starterbacteria, L. reuteri ATCC 55730 and S. thermophilusCECT 986. The defined oat formulation achieved astarter survival above the minimum level suggestedfor ensuring health benefits (107 cfu/ml) and so it canbe considered as a functional food. The starter viabilitywas maintained during the whole shelf life typical ofthis kind of product. The metabolic activity of the star-ters remained during cold storage due to the availabilityof nutrients, of which, as expected, monosaccharidesare mainly consumed. Thanks to their high stability, areasonable ratio of oat b-glucans remained in the finalfermented products which have a positive effect on theproduct matrix structure, due to their thickening andgelling properties. Besides the technological advan-tages, these compounds, together with the addedinulin, make the product one of great nutritional inter-est, since both of them are stated as prebiotics. Thus,consumers might benefit from both the nutritional andhealth properties of these functional food ingredients:probiotics and prebiotics. Therefore, a new synbioticnon-dairy milk was successfully developed in whichhigh probiotic survivals were assured throughout thetypical yoghurt-like shelf life.
FUNDING
This research was supported by the Universitat Politecnica deValencia (PAID-05-11-2740). This work was also supportedby the Conselleria de Educacion of Valencia government,which granted the author N. Bernat (ACIF/2011).
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