5. C ASHEW NUT FERMENTED PLANT - BASED « CHEESE » SUPPLEMENTED WITH RED SEAWEEDS
5.3 Results and discussion
5.3.4 Microbial load
Microbiological evaluation of CNFCAs is presented in Table 5.5 and p-values in Table E5.6 (annexes). The mean counts of Enterococcus exceed four Log CFU·g–1 for all samples and do not show significant differences between them (p>0.005).
Lactic Acid Bacteria (LAB) showed a significant difference between CNFCA-C vs.
CNFCA-CC and CNFCA-CC vs. CNFCA-P (p=0.0139). LAB predominated over other micro- biological groups, achieving the same counts for CNFCA-CC and CNFCA-P (5.48 CFU·g–1), and slightly exceeding by one Log CFU·g–1 for CNFCA-CC. In all case, the values obtained were higher than the limits referred by Saraiva et al. (2019). These elevated counts may be related to the addition of Advanced Acidophilus Plus (Solgar®), which contained Lactobacillus acidophilus LA-5® (250 million organisms), and Bifidobacterium lactis BB-12® (250 million or- ganisms). In fact, Lactobacillus is a genus with important applications in food fermentation, as it is also capable of producing lactic acid due to the metabolism of sugars (Swain et al., 2014).
Apart from that, LAB is responsible for the production of substances that can improve the flavor, texture, nutritional value, shelf-life, and safety of foods (Pisano et al., 2019).
Aerobic mesophilic bacteria (AMB) are the most represented group, ranging from 7.36 to 7.83 Log CFU·g–1. The statistical analyses revealed significant differences among all CNFCAs (p<0.05). Usually, cashew nut or cashew nut products present a higher AMB activity.
For example, Muniz et al. (2006) reported AMB counts (4.0 to 7.0 Log CFU·g–1) for cashew nut, whereas Göçer & Koptagel (2022) reported 8.84 ± 0.26 Log CFU·g–1 for a cashew nut-based
beverage fermented with kefir. Due to the high found level, and according to Saraiva et al. (2019), all CNFCAs are rated as unsatisfactory (>107 CFU·g–1) (see Table A2 annexes).
Table 5.5 — Levels of Enterococcus, Lactic Acid Bacteria (LAB), Aerobic Mesophylic Bacteria (AMB), Marine Agar Counts (MAC), Glucose-Yeast-Peptone (GYP) molds and yeasts, Escherichia coli, Total Coliforms (TC), Staphylococ- cus aureus, Salmonella spp., and Listeria monocytogenes for cashew nut fermented chese alternatives (CNFCAs):
cashew nut fermented cheese alternative control (CNFCA-C); cashew nut fermented cheese alternative with Chon- drus crispus (CNFCA-CC); and cashew nut fermented cheese alternative with Porphyra sp. (CNFCA-P) [a].
Microbiological parameters (Log CFU·g–1)
Supplemented products CNFCA-C CNFCA-CC CNFCA-P
Enterococcus 4.48 ± 0.00 4.48 ± 0.00 4.48 ± 0.00
LAB 6.56 ± 0.00 5.48 ± 0.00 5.48 ± 0.00
AMB 7.83 ± 0.86 7.36 ± 0.80 7.48 ± 0.00
MAC <2 6.18 ± 1.63 6.08 ± 0.18
GYP (Molds) <2 <2 <2
GYP (Yeasts) 3.76 ± 1.64 <2 <2
E. coli 2.11 ± 0.33 <1 1.9 ± 1.89
TC 0.7 ± 0.15 0.7 ± 0.15 2.57 ± 0.00
S. aureus 2.18 ± 1.63 2.54 ± 0.37 2.72 ± 0.42 Salmonella spp. Abs. in 25 g Abs. in 25 g Abs. in 25 g L. monocytogenes Abs. in 25 g Abs. in 25 g Abs. in 25 g
[a] Counts are expressed as Log CFU·g–1 and data as mean ± SD (n=4).
Count on marine agar achieved similar values for CNFCA-CC (6.18 Log CFU·g–1) and CNFCA-P (6.08 Log CFU·g–1). Cashew nut fermented cheese alternative control (CNFCA-C) does not show microbiological activity (<2 Log CFU·g–1). The statistical analyses revealed sig- nificant differences between CNFCA-C vs. CNFCA-CC and CNFCA-C vs. CNFCA-P (p=0.0139). A plausible justification for this is that the values for the supplemented products are due to seaweeds. However, in chapter 2, C. crispus and Porphyra sp. had an activity of 4.9 Log CFU·g–1 and 4.7 Log CFU·g–1, respectively.
Molds were deemed satisfactory, presenting the same values for all samples (<2 Log CFU·g–1), and do not showing significant differences between them (p>0.05). Some authors suggest that contamination of cashew nuts by molds can occur early in the field or during a prolonged storage time (Schmitt et al., 2018), which was not the case. At same time, the previ- ous blanching of cashew nuts in hot water helps to destroy microorganisms such as bacteria, yeasts, and molds (Renard & Maingonnat, 2020).
Concerning yeasts, CNFCA-CC, and CNFCA-P register <2 Log CFU·g–1, whereas CNFCA-C exceeded 3.0 Log units, showing statistical differences between CNFCA-C vs.
CNFCA-CC and CNFCA-C vs. CNFCA-P (p=0.0139). The results indicated that seaweeds could inhibit yeast’s activity, which is not surprising since some seaweeds demonstrate anti- fungal capacity against strain yeasts (Pérez et al., 2016).
Results of Escherichia coli ranged from <1 Log CFU·g–1 to 2.11 Log CFU·g–1, showing sta- tistical differences among CNFCA-C vs. CNFCA-CC and CNFCA-CC vs. CNFCA-P (p=0.0126).
Mendes et al. (2013) demonstrate that ethyl acetate and diethylester extracts of C. crispus from wild and from an IMTA system possess antimicrobial activity for against the growth of bacte- ria such as E. coli and others. Total coliforms (TC) ranged from 0.7 to 2.57 Log CFU·g–1, reveal- ing differences among CNFCA-C vs. CNFCA-P and CNFCA-CC vs. CNFCA-P (p=0.0126).
CNFCA-P shows a high content of total coliforms; however, these results are not linear with the found values (see chapter 2) for C. crispus (3.0 Log CFU·g–1) and Porphyra sp. (2.0 Log CFU·g–1).
Counts of coagulase-positive S. aureus reached similar counts between samples, ranging from 2.18 to 2.72 Log CFU·g–1. The statistical analyses revealed significant differences among CNFCA-C vs. CNFCA-P (p=0.0194). The low values for S. aureus were considered satisfactory from a safety point of view since the production of toxins only occurs at higher counts (>104) (HPA, 2009).
Salmonella spp. was absent through the plating count (not present per 25 g). In fact, a US surveillance study shows that the prevalence of Salmonella (95% CI) in cashew nuts was mini- mal (0.55%), i.e., occurred in just 4 of 733 samples (Zhang et al., 2017).
Listeria monocytogenes was also absent through the plating count (not present per 25 g).
As reported by Eglezos (2010) «there are no data available on the prevalence of L. monocyto- genes in cooked edible nut kernels or any foodborne illness lined to the presence of L. mono- cytogenes in this kind of product». On the other hand, LAB, which is one of the best represented groups in the CNFCAs (5.48-6.56 Log CFU·g–1), has been demonstrated bactericidal and bac- teriostatic properties against foodborne pathogens such as Salmonella spp. and L. monocyto- genes (Chen et al., 2022).
5.3.5 Flash profile methodology
Results obtained in the second session of the Flash Profile from the 8 panelists ranking for the CNFCAs (CNFCA-C, CNFCA-CC, and CNFCA-P) per attribute, according to the final list of terms defined, were analyzed by Generalized Procruste Analysis (GPA). Samples that presented the lowest residual variance for the analyzed attributes had the most consensual rankings. Therefore, CNFCA-C was the most consensual as it presents the lowest residual var- iance for all attributes, except for texture, for which it presents the highest percentage (20.6%).
Cashew nut fermented cheese alternative with C. crispus (CNFCA-CC) had the highest resid- ual variances for appearance (29.9%), flavor (18.1%), and after-taste (22.3%), while CNFCA-P had the highest residual variance for aroma (17.2%), showing that the supplementation of sea- weeds affects the consensus and influences the sensory characteristics of products, certainly due to the sensory complexity of the seaweeds (see Table 5.6).
Table 5.6 — Residual variance values for each CNFCAs from Generalyzed Procrustes Analysis (GPA) of FP.
Attributes Object Residual (%)
CNFCA-C 12.412
Appearance CNFCA-CC 29.920
CNFCA-P 13.578
CNFCA-C 9.729
Aroma CNFCA-CC 11.096
CNFCA-P 17.230
CNFCA-C 6.051
Flavor CNFCA-CC 18.133
CNFCA-P 13.764
CNFCA-C 20.604
Texture CNFCA-CC 16.473
CNFCA-P 15.811
CNFCA-C 7.781
After-taste CNFCA-CC 22.348
CNFCA-P 12.737
Residual variances values for each panelist calculated by GPA reveal the panelists hav- ing higher residual variance values: a higher percentage for appearance for panelist 7 (14.1%), aroma for panelist 8 (10.2%), flavor for panelist 1 (15.0%), texture for panelist 4 (22.2%), and after-taste for panelist 1 (13.4%), which indicates that these panelists were further from the consensus (Table 5.7).
The values of consensus index (Rc) (Table 5.8) were as follow: appearance (33.8%), aroma (31.2%), flavor (29.1%), texture (11.6%), and after-taste (23.4%). All attributes show an inade- quate consensus in the performance of the panelists, particularly on texture and after-taste.
These values were lower when compared with Hernández-Cervantes et al. (2010) in the evaluation of a Cuajada-type cheese, where Rc=0.553 (55.3%).
Figure 5.3 shows the coordinates of objects (CNFCAs) after GPA analysis and the corre- lations between the attributes (appearance, aroma, flavor, texture, and after-taste), and the di- mensions F1 (first axis) and F2 (second axis).
Table 5.7 — Residual variance values, scaling factors, and the percentage variation explained by the first two prin- cipal components (F1 and F2) of Generalyzed Procrustes Analysis (GPA) for each panelist from the cashew nut fermented cheese alternatives (CNFCAs) flash profile analysis.
Attributes Panelists Residuals Scaling factors F1 (%) [a] F2 (%) [a]
1 3.035 1.273 88.209 11.791
2 3.052 1.052 69.690 30.310
3 12.927 0.846 56.997 43.003
Appearance 4 3.139 0.807 91.871 8.129
5 0.778 1.084 82.048 17.952
6 5.608 0.856 73.117 26.883
7 14.102 1.434 97.387 2.613
8 13.270 1.110 86.214 13.786
1 0.498 1.870 66.932 33.068
2 3.683 1.018 87.487 12.513
3 4.850 0.849 47.790 52.210
Aroma 4 9.474 1.051 96.713 3.287
5 5.055 1.464 69.484 30.516
6 2.233 0.711 78.478 21.522
7 2.017 0.933 73.758 26.242
8 10.246 1.090 32.856 67.144
1 15.007 1.343 63.159 36.841
2 2.427 1.071 85.080 14.920
3 1.550 0.870 71.391 28.609
Flavor 4 3.695 0.875 65.392 34.608
5 2.253 1.199 84.965 15.035
6 6.496 0.661 96.886 3.114
7 3.960 1.799 93.687 6.313
8 2.560 1.247 78.470 21.530
1 2.255 2.892 81.355 18.645
2 4.433 0.816 77.693 22.307
3 2.929 0.831 62.728 37.272
4 22.185 0.000 0.000 0.000
Texture 5 3.795 2.562 85.668 14.332
6 4.479 0.752 95.204 4.796
7 3.843 0.748 60.659 39.341
8 8.968 3.992 13.658 86.342
1 13.893 1.355 61.013 38.987
2 0.763 2.262 88.815 11.185
3 4.111 1.011 62.999 37.001
4 3.831 0.673 65.162 34.838
After taste 5 4.587 1.108 91.752 8.248
6 4.378 0.641 96.684 3.316
7 3.337 1.709 97.119 2.881
8 7.966 1.578 94.090 5.910
[a] F1 (1st principal component of GPA), and F2 (2nd principal component of GPA).
For the appearance attribute (a), the CNFCA-C is perceived as cohesive, porous, and with holes, whereas CNFCA-CC as brownish, greasy, purple, rose/rosy, and soft, and CNFCA- P as opaque. Although there is a discrepancy between the terms used to describe CNFCA-C, the terms porous and with holes are in line with the findings, where CNFCA-C had reached the lowest values for hardness (6.01 N) and cohesiveness (0.33). CNFCA-CC is described by terms such as purple, rose/rosy and pink seaweeds, which correspond with the found value (3.19) for color parameter a* (degree of redness, a*>0, or greenness, a*<0). The opacity described in CNFCA-P is in concordance with L* parameter (degree of lightness, L*=0, meaning dark, and L*=100 white) and chroma (C*) since that in both cases present the lowest values, namely 38.30, and 15.20, respectively.
In the attribute aroma (b), CNFCA-C was mainly characterized by cheese, dried fruits, flamengo cheese, fermented, and yoghurt, whereas CNFCA-CC by cigar, nam pla, salty, straw of coffee, and wood, and CNFCA-P by dry herbs. The aroma of dried fruits attributed to CNFCA-C is desirable since dried fruit is in the base of the product. An analog term, nutty, was reported by Lima et al. (2012) to a cashew nut butter made from different kernel grades quality. At the same time, terms such as cheese, flamengo cheese, fermented or even yoghurt suggest a similitude between CNFCA-C and dairy cheese, due to the addition of Lactobacillus acidophilus LA-5® (Advanced Acidophilus Plus, Solgar®). In fact, CNFCA-C is the sample that shows the higher count of LAB (6.56).
None of the descriptors for supplemented products can be associated to volatile organic compounds. However, dry herbs described for CNFCA-P can be associated with green odor, which are related to esters (Vilar et al., 2020).
Table 5.8 — Consensus index (Rc (%)) among the panelists for each attribute (appearance, aroma, flavor, texture, and after-taste) of cashew nut fermented chese alternatives (CNFCAs).
Attributes Rc (%)
Appearance 33.8%
Aroma 31.2%
Flavor 29.1%
Texture 11.6%
After-taste 23.4%
Results for flavor (c) show that CNFCA-C was mainly characterized by bread and nau- seating; CNFCA-CC by sour; and CNFCA-P by Dulse, Nori, and spices. Regarding the term nauseating described for CNFCA-C, tree nuts are a common cause of allergy, and their symp- toms typically occur within minutes of ingestion, ranging from medium to severe symptoms, such as anaphylaxis. Among several symptoms, they can include abdominal pain, cramps, nausea, and vomiting (Thermo Fisher, 2022), and, as such, a possible relationship cannot be ruled out.
Figure 5.3 — Biplot map of Generalized Procrustes Analysis (GPA) performed on Flash Profile (FP) data and the lexicon used to describe the various attributes of the CNFCAs at the F1 and F2 dimensions: a) appearance; b) aroma;
c) flavor; d) texture; and e) after-taste.
It can be inferred that some of the panelists, despite being trained, confuse the flavor of seaweeds, since CNFCA-P was described as Dulse (Palmaria palmata) and Nori (Porphyra sp.);
something that is perfectly understandable, since sometime aromas and flavours can be very complex and difficult to distinguish from others. The other term attributed to CNFCA-P,
‘spices’, are related to carboxylic acids (Vilar et al., 2020).
Concerning texture (d), CNFCA-C was mainly characterized by cohesiveness, hardness and adhesiveness, whereas CNFCA-CC by terms such as arenaceous, dry, hardness of parti- cles, and light, and CNFCA-P by brittle. The cohesivity described for CNFCA-C agrees with the value obtained in this study (0.33). Dry and brittle terms described for CNFCA-CC and CNFCA-P also comply with the founded hardness values, namely 7.90 and 9.69 N, respec- tively. Particles hardness for CNFCA-CC can be attributed to the texture of C. crispus, which is generally firm, or cartilaginous-like (Figueroa et al., 2022). On the other hand, the size of the particles (2.87 mm) may also have influenced the sensation described.
For after-taste (e), CNFCA-C is characterized by several terms such as aromatic persis- tence (temporal), lactic fat, and dry fruits, whereas CNFCA-CC by acid and rancid, and CNFCA-P by bitter, Dulse, Nori, smoked, and sour.
During the fermentation process, lactic acid is produced through sugar metabolism, which is responsible for the increasing sourness as the sweetness reduced (Swain et al., 2014).
In general, the supplementation of food products with seaweeds compromises their sensory
attributes due to the appearance of off-flavors (Afonso et al., 2019), for example, rancidity, as described for CNFCA-CC. This rancidity can be attributed to aldehydes, including heptanal and octanal, which contribute to undesirable rancid odour and flavour during spoilage of fat and fatty foods (Giri et al., 2010; Peinado et al., 2014). Also, aldehydes such as heptanal and 2- octenal are associated with ‘smoked’ and ‘sour’ (Peinado et al., 2014), respectively, both at- tributed to CNFCA-P.
In summary, seaweeds have a decisive role in various attributes, especially aroma, fla- vour, and after-taste, which in fact changed the descriptions of all the attributes making it possible to clearly distinguish CNFCAs.
Conclusions and future perspectives
The cashew nut fermented cheese alternatives can be seen as an interesting alternative to dairy cheese consumption as they provide an overall nutritionally healthier profile. The supplementation with seaweeds influenced the technological/sensory characteristics of the CNFCAs and thus contributes to confer different properties to the «cheeses», enhancing di- versity and producing quality products. Seaweeds influenced positively parameters such as total solids, and ash, which show good values for the supplemented products. On the other hand, total lipids and crude protein content show lower values. Macro-and micro minerals increase when it was added C. crispus to the curd, namely Ca, K, Mg, Na, Fe, I, Se, and Zn. The instrumental color and some of the textural parameters (hardness, springiness, and gummi- ness) were significantly influenced by the addition of seaweeds to the food matrix. Concerning to the microbiota, all parameters comply with the European guidelines, except for AMB in all samples and for MAC in both supplemented products. Furthermore, the data obtained suggest the presence of a spontaneous lactic fermentation possibly due to the addition of capsules con- taining Lactobacillus acidophilus, which was responsible for the production of substances that can improve several aspects of products (e.g. flavor, texture, nutrition, shelf-life, and hygienic quality).
The supplemented products had very distinguishable characteristics, and a good ac- ceptance by pannelists participating in the Flah Profile sessions, having high potential to con- tribute to increase diversity of products available for consumers that require alternatives to dairy products, as well as allowing the familiarization of consumers with seaweeds.
Further studies are necessary to improve the manufacturing process, to increase the time of fermentation, improve the texture and PC of the products. Other aspects include developing
«cheeses» based in new nut pastes and/or introducing other seaweeds.
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