Protein estimation and statistical analyses

Protein estimation were conducted using the methods in chapter 3.4.1 and flow chart in figure 3.5 and and statistical analyses using the methods in chapter 4.2.3. The percentage unbound fraction was calculated from equation 6.1 where;

Equation 6.1

( ) Where a = recovered protein inTris pH 7.4 (µg)

b = weight of lypholized sample used (µg)

Percentage bound fraction was calculated from equation 6.2 where;

Equation 6.2

( ) Where c = recovered protein in NaOH (µg)

b = weight of lypholized sample used (µg) Total protein (TP) was calculated from equation 6.3 where;

Equation 6.3

Where T = unbound fraction

N = bound fraction

The percentage of protein in wet macroalgae (PW) in equation 6.4 was calculated using the % lypholized weight from equation 4.1.

Equation 6.4

(

) Where d = % lypholized weight

6.3 Results

Figures for the mean protein ( converted to mg g^-1) content recovered from the lypholized macroalgae can be found in appendix 6.2. Results from the ANOVA and general linear model for species by year season and month are shown in appendix 6.3. Graphical representations of the monthly variation of total protein recovered can be seen in appendix 6.4.and in appendix 6.5 the l

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mean monthly (unbound) protein µg g^-1 recovered from lypholized macroalgae in Tris pH 7.4 buffer by month of collection.

The ANOVA (table 6.1) shows a significant influence of both and species (P<0.01) and month of collection (P<0.01). The principal source of variability is the species and it is therefore acceptable to treat the individual species as independent. Each species was subjected to ANOVA General linear model and post – hoc Tukey analysis to examine the influence of month of collection on the variables. Probability statistics for the analyses are shown in table 6.2.

Table 6.1 Analysis of variance for total percentage protein recovered, using Adjusted SS for tests with species and month of sampling as the model.

Analysis of Variance for total mg g^-1, using Adjusted SS for Tests Source DF Seq SS Adj SS Adj MS F P Species 8 511438 512748 64093 73.18 P<0.01 Month 23 62748 62748 2728 3.11 P<0.01 Error 614 537749 537749 876

Total 645 1111934

S = 29.5941 R-Sq = 51.64% R-Sq(adj) = 49.20%

There was no effect of the year on the overall means of bound, unbound and total protein as can be seen in table 6.2 but there was an effect (P<0.01) when the protein recoverable from PW was considered. The PW differs between years with July 2009 – June 2010 (3.8 %, year 1 ) having a higher recovered percentage protein than July 2010 – June 2011 (3.3 %, year 2).

For individual species, there was an effect of the month of collection on the bound protein recovered in all species except FV. For the unbound protein, there was an effect of the month of collection for species AN, FV, LH and UL. In all other species, there was no significant effect. In the case of total protein recovered there was no effect of the month of collection on AN and FS, in all other cases there was an effect (P<0.05) of the month of collection on the results.

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Table 6.2 Probability statistics for effects of Year, Species and Month of collection on bound and unbound protein recovered from lypholized macroalgae samples where: TP = Total Protein and PW = calculated percentage protein in the wet sample.

153 6.3.1 Total percentage proteins

The mean percentage total protein recovered for individual species is shown in figure 6.1. Both PU (160, SE 6.06 mg g^-1; 22 %) and FV (208, SE 6.06 mg g^-1; 21 %) have the highest recovered protein levels and are not significantly different from each other. The lowest mean percentage protein recovered is MS (13.4, SE 3.09 mg g^-1; 13.4 %). Differences in the maxima and minima for total percentage monthly recoveries are 4.9, 4.2, 6.1, 9.4, 6.4, 7.9, 7.6, 17.7, and 12.2 % for AN, FS, FV, LD, LH, MS, PP, PU and UL respectively.

Figure 6.1 Total percentage protein, bound plus unbound protein recovered from lyophilized macroalgae. Where; AN, FS, FV, LD, LH, MS, PP, PU and UL represent Ascophyllum nodosum, Fucus serratus, F. vesiculosis, Laminaria digitata, L. hyperborea, Mastocarpus stellatus, Palmaria palmata, Ulva lactuca and Porphyra umbilicalis respectively.

6.3.2 Results for Ascophyllum nodosum (AN)

The overall mean percentage was 16.4 % (figure 6.1). In the analysis of total protein, although there was a difference between the years (figure 6.2) the monthly Tukey analysis indicated there was no significant difference between months or season of collection. In the case of the unbound protein, July 2009 had a significantly lower quantity of unbound protein recovered than June 2011

0 5 10 15 20 25

AN FS FV LD LH MS PP PU UL

Total Percentqge Protein Recovered

Species

% Protein

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(0.3 vs. 1.6 mg g^-1, P<0.05) but all the other months were not significantly different. For PW this is reversed and July 2009 has the highest recovery (13.5

%). There does not appear to be a strong seasonal component in the protein recoverable from AN (figure 6.3) and the seasonal effect in the PW (figure 6.4) is driven by the result of July 2009.

Figure 6.2 Total percentage protein recovered by species and year (July 2009 – June 2010, year 1; July 2010 – June 2011, year 2) of collection.

0 5 10 15 20 25 30 35

1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2

AN FS FV LD LH MS PP PU UL

Total Percentage Protein in Lyophilized Macroalgae

Species by Year Total %

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Figure 6.3 Total percentage protein recovered from lypholized macroalgae by species and season of collection

0 5 10 15 20 25 30

Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter

AN FS FV LD LH MS PP PU UL

Total Percentage Protein Recovered

Species by Season total % protein

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Figure 6.4 Estimated total percentage protein recovered from wet macroalgae by species and season of collection.

0 1 2 3 4 5 6 7

Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter Autumn Spring Summer Winter

AN FS FV LD LH MS PP PU UL

Percentage Protein in Wet Macroalgae

Species by Season

%Protein in Wet

157 6.3.3 Results for Fucus serratus (FS)

For FS the overall mean recovered was 15.4 % (figure 6.1) with no effect of the year of collection (figure 6.2). In the seasonal (figure 6.3) and monthly means, there are no significant differences in the bound, unbound or total protein. The monthly range is 17.8 to 13.6 % protein. In the PW for FS although significant differences occur between months there does not appear to be any seasonal pattern (figure 6.4).

6.3.4 Results for Fucus vesiculosis (FV)

In figure 6.1, the overall mean protein recovered was 20.8 % for FV and again there is no effect of the year of collection (figure 6.2). The total monthly protein recovered the maximum is 23.8 %, Dec 2009 and the minimum 17.7 %, Feb 2010. However, the Tukey analysis does not indicate that any one month is statistically different. In the results for PW in figure 6.4 the Tukey analysis

indicates a peak percentage protein is found in summer but the peak recovered is in April 2010 (23.1 %) and declines until Feb 2011 peaking again in March 2010 (23.7 %).

6.3.5 Results for Laminaria digitata (LD)

The overall mean of 16.0 % protein for LD is in figure 6.1. The maximum recovered as 20.6 %, Jan 2011 and the minimum 11.2 %, June 2011. There is no effect of the year of collection (table 6.2, figure 6.2 except in the estimated protein in PW. This shows statistical differences for the year season and

month of collection (P<0.01) the Tukey analysis indicates that a month one year can be one of the lowest in the subsequent year e.g. December 2009 (2.05 %) compared to December 2010 (1.38 %).

6.3.6 Results for Laminaria hyperborea (LH)

The results for LH in figure 6.1 show an overall mean protein percentage of 14.4 % and an effect of the year of collection (table 6.2, figure 6.2). The monthly analysis has a maximum of 16.8 % ( Jan 2010) and a minimum 10.4 % (Aug 2009) but the Tukey analysis does not indicate that there is any statistical

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difference between months. In the PW (figure 6.4) although autumn has the highest PW, the lowest estimate percentage proteins occur when the levels drop in March 2010 (3 %) and March 2011(1.8 %), Sept 2010 (2.4 %), May 2011 (2.2 %) and highs in the winter months, Jan 2010 (2.5 %) and Jan 2011(3.8 %).

6.3.7 Results for Mastocarpus stellatus (MS)

In figure 6.1, the overall mean protein recovered for MS is 13.4 %. The monthly results show a maximum of 18.9 % June 2011 and minimum of 11.0 %, Dec 2010. There is an effect of the season of collection in the protein; bound, total and PW. In the PW statistically significant peaks appear in March and April 2010 (6.3 and 6.2 %) and again slightly later next year in April and May(4.4 and 5.4 %) and are reflected in the higher peak for spring in figures 6.3 and 6.4.

6.3.8 Results for Palmaria palmata (PP)

In figure 6.1 PP shows an overall mean of 19.7 % protein and in figure 6.8 the maximum is 22.4 % in May 2011 and the minimum in Feb 2010 of 14.8

%. There are significant changes over 3 – 4 month cycles but they do not appear to be driven by the season as seen by Aug 2009 (having one of the lowest measurements (16.3 %) and Aug 2010 one of the highest (22.5 %).

6.3.9 Results for Porphyra umbilicalis (PU)

The overall mean for PU (figure 6.1) is 22.0 %. In figure 6.5 it can be seen there is a maximum of 33.6 % protein recovered in Feb 2011 and a minimum 15.9 % in Feb 2010. The highest percentage recovered protein is in late winter March 2010 (29.5 %) and Feb 2011(33.6 %) with reductions from May to Sept in all years. This pattern is also seen in the percentage protein recovered from the wet weight. This also corresponds with the Tukey analysis, which indicates that the protein levels found in autumn (23.5 %) and winter (26.2%) are higher than spring (19.1 %) and summer (19.6 %) as seen previously in figure 6.3 and 6.4.

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Figure 6.5 Total percentage protein extracted from lypholized PU and calculated percentage recoverable from wet PU by month of sampling.

0 5 10 15 20 25 30 35 40

Percentage Protein Recovered

Month of Sampling

PU%dry PU %Wet

160 6.3.10 Results for Ulva lactuca (UL)

In figure 6.1 UL shows an overall mean protein percentage of 16.2 %.

There appears to be an effect of the season and month of collection (table 6.2) with a maximum of 23.9 % in Feb 2010 and a minimum of 11.7 % in May 2011.

Figures 6.3 and 6.4 show a high of total protein recovered and PW in winter.