New Antioxidant Ingredients From Brewery By-Products For Cosmetic Formulations 2

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2.11. Statistical Analysis

Data are shown as mean ± standard deviation (SD) of at least three independent experiments. Statistical analysis was performed using one-way ANOVA with Dunnett or Bonferroni post hoc test and Student's t-test, as appropriate. Differences were considered significant at p<0.05. Analyses were performed using GraphPad PRISM software (version 5.0; GraphPad Software, La Jolla, CA, USA) on a Windows platform.

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3. Results and Discussion

3.1.Brewing Process of Craft Beers under Study

Craft beers, unlike industrially produced beers, are not pasteurized or filtered, and thus preserve more of their composition, aroma, and taste. The composition of craft beers is simply water, malt, hops, and yeast (Saccharomyces Cerevisiae), with no other additives, and thus the chemical ingredients present in the beer depend on the ingredients that are added and removed during the brewing process [8]. Craft beer producers generally avoid adding citric acid, which can contribute to reduced product oxidation, or other additives such as aroma, sugars, flavors, and juices [8]. In the beers studied in the present work, no additives were used and the components were unprocessed water, malt, hops, and yeast.

Table 1 lists the beers studied in the present work and provides their composition and main characteristics. Some other information about the beers is confidential and thus could not be disclosed. Figure 1 illustrates the brewing process of craft beers used for this work.

The craft beer brewing process begins with the mixture of malt and water in appropriate proportions. Five different malts can be used and mixed together according to different recipes(Table 1). Water and malt are heated at a temperature of 70°C for 90 min and the resulting wort is filtered to remove the spent malt. In the next step, two different hops, Perle and Saaz, can be used in different proportions. The hops are added to the filtered wort and boiled at 100°C for 90 min, after which the spent hops are removed by centrifugation (Whirpool process) at an interval of 1300-1550, depending on the batch size. The subsequent step is fermentation when Saccharomyces Cerevisiae yeast is added and heated at 20-22°C for 90 min to convert the sugars into alcohol. The spent yeast is then removed by centrifugation, the resulting beer is bottled, and, after a variable period of maturation of 20-30 days, is ready for consumption. To summarise, the brewing ingredients are water, malt, hops, and yeast. The intermediate products are wort, wort after hops(the wort after boiling with hops and subsequent removal of the spent hops), and the beer after yeast(the beer formed after fermentation and subsequent removal of the spent yeast). The final product, of course, is the matured beer. The spent materials are malt, hops, and yeast. All these products were fully analyzed for total phenol content and antioxidant capacity.

3.2. Determination of Total Phenol Content

A previous study[10] identified forty-seven polyphenols in four types of commercial beers， namely， lager， Pilsen， Märzebier, and non-alcoholic beer， by using an electrospray ionization hybrid linear ion trap quadrupole Orbitrap mass spectrometry technique. Among the polyphenols, it is possible to list phenolic acids, hydroxycinnamoylquinics, flavonols, flavones, alkylmethpxyphenols, alpha- and iso-alpha-acids, hydroxyphenyl acetic acids, and prenylflavonoids.

In craft beers, another study identified phenolic and nitrogenous compounds by high-performance liquid chromatography and mass spectrometry [11]. Fifty-seven phenol compounds were identified, together with eleven nitrogenous compounds belonging to the phenoxide class.

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In our previous study [12], twenty phenol compounds, for example, gallic acid, catechin, or humulone, were quantified in the same six types of craft beers, worts, ingredients, and spent products of the present study by a validated LC-MS/MS method. The sum of phenol compounds (SPC) identified and quantified in barley malts was non-negligible and was prevalently due to trans-p-coumaric acid, which was transferred to the worts during the must preparation and was responsible for the non-negligible SPC of worts. Bitter acids and prenylflavonoids were detected in the starting hops, while their concentration decreased in the spent hops, suggesting that they were transferred to the intermediate of production. Phenolic compounds, largely present in starting barley malts and hops, decreased in the final beers because they were absorbed into the yeast added for the fermentation.

Based on these previous results, one can surmise that the phenol compounds may influence the Total Phenol Contents (TPC) of the beers under study. Looking at the results of our TPC analyses, reported in Table3, it seems that the extraction solvent has a strong bearing on the TPC. In fact, starting malts subjected to ethanol extraction showed higher TPC values than those subjected to water extraction, with quite a large range of values for extract in ethanol, from 28 to 72 mg GAE/g, and a more limited range for extract in water, from approximately 11 to 16 mg GAE/g. cistanche life extension This indicates that for the compounds that can influence the TPCin this study, extraction in ethanol is more effective than that in water. Similar results regarding TPC values were reported by Zhao et al. [9] for 14 varieties of barley subjected to extraction in acetone, yielding values from 2.17 to 2.56 mg GAE/g. Thus, in the work of Zhao et al. (2008) acetone appeared less effective at extracting phenol compounds from barley than water or ethanol 70°in this study. Several other studies have also demonstrated that ethanol is effective at extracting compounds that influence the TPC [24,25]. Our data on the starting malt types indicate that Type 3 and 5 malts have a higher TPC than the other ones(Table 1) because they are present when values are the highest.

Regarding the TPC of wort, it should be borne in mind that this product was not subjected to extraction, but used as received from the brewery. The TPC of wort was lower than that of starting malt and depends on the first brew phase, which consists of heating malt and water at a temperature of70°Cfor 90 min. During this phase, phenols can diffuse from the coarse grains (malt grains are only coarsely ground) and dissolve into the wort. However, once received by us, starting malt was milled to recover fine particles to optimize the phenol extraction. cistanche nz This can explain the highest value of starting malt appearing with respect to wort: phenols can only partially be released from coarse particles during wort production, and phenols that are still inside the grains can be easily released from finest particles during extraction in water or in ethanol 70°.

The spent malts exhibited intermediate values between those of the starting malt and the corresponding worts, confirming that phenolic compounds were still present in spent malt: the extraction in water and ethanol 70°revealed appreciable TPC values ranging from approximately 9 to 14 mg GAE/g, and from 12 to 37 mg GAE/g, for extraction in water and ethanol, respectively.

Table 3 reports the TPC values of both pure hops Perle and Saaz. Both starting hops showed a very high TPC, and the values obtained after extraction in ethanol were again higher than those obtained in water, confirming ethanol as a better solvent than water for the extraction of phenols. The Perle starting hops showed a higher value than the Saaz one. Nevertheless, they were not used as pure hops but mixed according to a secret recipe. Thus, the mix used for every brewing process was analyzed. The TPC can correspond to the mix of the two different hops in various percentages, which is approximately intermediate between the percentage of pure hops. The TPC of worts obtained after the addition of hops was higher than that of worts before the addition of hops, which indicates that part of the phenolic compounds is transferred from the hops to the wort during the brewing process, which, in this phase, consisted of boiling hops in wort at 100°C for 90 min. Nonetheless, despite the very high TPC of hops, the TPC of worts showed a modest increase. One might expect that the spent hops would have a high TPC, but the TPC was actually lower, which probably indicates that a large part of the phenolic compounds was lost during the process, due to the thermal instability of some phenolic compounds [26].

The starting yeast showed an appreciable TPC, particularly when the extraction was carried out in the water, while a far lower value was obtained from extraction with 70°ethanol. This can be explained by the fact that pure yeast is less soluble and less hydrated in ethanol than in water, and thus the extraction is less efficient. cistanche penis size It appears that part of the TPC in yeast was transferred to the beer, as there was an increase in the TPC of the corresponding beers. Again, it should be noted that the analysis was performed on beers that were not subjected to extraction, which were thus not influenced by the extraction method. However, the TPC of spent yeasts is of particular interest because it was non-negligible. In fact, the TPC of spent yeasts after water extraction was slightly lower than that of starting yeasts, while the values for spent yeasts after extraction in ethanol were even higher than those in starting yeasts. This is due to the hydration of the yeast during fermentation, which favored the dissolution and extraction of phenols.

The TPC of the final beers was not statistically different (p<0.05) from that of beers after yeast, indicating that the compounds remain stable during beer maturation.

To summarise, the final beers were enriched with phenolic compounds throughout the brewing process, during which the various ingredients transferred these compounds to the beer. The highest TPC was found in the Triplo Malto and the Maior beers. Waste was only partially exploited and non-negligible TPC values were highlighted and were particularly significant for yeast when extraction was carried out in the water.

3.3. Evaluation of the Antioxidant Activities

The antioxidant activities were evaluated by assessing the Trolox equivalent antioxidant capacity (DPPH),ferric-ion-reducing antioxidant parameter (FRAP), and radical cation scavenging activity and reducing power(ABTS), and respective results are reported in Tables 4-6.

PDF for starting malt ranged from approximately 9 to 24 μmol TE/g for water extracts and from 20 to 42 mol TE/g for ethanol extracts. The DPPH values were generally higher than the values obtained by Zhao et al. [9] after the extraction of acetone. They actually reported that the radical scavenging activities of 14 samples of malts ranged from 9.33 to 11.78 μumol TE/g.

Regarding wort, one must note that values were the same for water and ethanol extraction. As explained before, the wort was not subjected to extraction and was provided as a solution by the brewery. The wort of different malts exhibited lower values than those of the corresponding starting malts. The reason for this could be the same as that explained for TPC, that is, an incomplete dissolution of molecules from malt to wort during the brewing process. The spent malts obtained after ethanol extractions exhibited higher values than those obtained after water extraction, but far lower values than those of starting malt. This means that some molecules were transferred to the wort, while others were lost during the process.

Both Perle and Saaz hops showed high DPPH values, particularly when extraction was carried out in ethanol (approximately 72-89 umol TE/g after extraction in water, and 258-354 μmol TE/g after extraction in ethanol）. Their mixes showed values that corresponded to the specific recipe used to produce each beer. The starting hops mix reflected the hops' composition.

Wort after the addition of the hops showed slightly increased DPPH values compared to the previous wort, meaning that some molecules influencing DPPH value transferred to the wort, but if one considers the strong decrease in DPPH values for spent hops, it is possible to conclude that the molecules influencing DPPH were destroyed during this brewing stage because they were thermally unstable [26]. Spent hops showed a very important decrease in DPPH values with the respect to starting hops, confirming the thermal instability of molecules influencing the DPPH value. cistanche powder Starting yeast exhibited modest DPPH values for extracts in water and in ethanol. It was interesting to note an increase in the wort after fast, and particularly in spent yeast, where a significant increase in DPPH values could be observed. The explanation can be found in the enzymatic reaction that occurred in presence of the yeast on flavonol glycosides: the enzymes of the yeast are able to convert glycosides to aglycones that are more reactant than the corresponding glycosides [27,28]. The DPPH values for final beers were not statistically different (p<0.05)from those of wort after yeast.

The antioxidant activity determined by the ABTS of starting malt ranged from approximately 21 to 47 μmol TE/g for extraction in water and from 41 to 97 for ethanol extracts, values higher than those determined by Zhao et al. [9]. Our findings are in good agreement with the observation of higher TPC values when extraction was carried out in ethanol. The starting malt type 5, which was only used for the production of Maior beer, had particularly high values. In the case of worts, the ABTS values were higher than those of corresponding starting malts; this indicates that the wort production process is able to extract more molecules that can influence the ABTS result, as was demonstrated for DPPH values. Spent malts exhibited lower ABTS values lower than those of starting malt, confirming that molecules are transferred to the wort during the process. The ABTS for starting hops was very high but decreased strongly in wort after hops. Residual molecules able to influence ABTS were present in the spent hop. The ABTS of starting veast was higher when extraction was carried out in the water, confirming the previous observation, that is, a better solubility of yeast in water than in ethanol. Beer after yeast showed high ABTS values, while spent yeast exhibited lower values, very similar to those of final beers. Antioxidant activity was then evaluated by FRAP. Starting malt showed values from 56 to 80 umol TE/g for water extracts， and from 33 to 54 μmol TE/g for 70°ethanol extracts. For water extracts, the highest value was that of Ego, while for ethanol extracts, the highest one was that of Alter. Worts exhibited lower values than starting malts, and no significant differences were highlighted among the different types. Spent malts did not show significant differences from starting malt. cistanche salsa extract Values for starting hops were nearly 332 and 377 umol TE/g for Perle and Saaz, respectively, when extraction was carried out in the water, while they were significantly lower,120and 110 umol TE/g, for Perle and Saaz, respectively, when extraction carried out in 70°ethanol, confirming the differences between the two extraction methods. This was also confirmed in the starting mixtures, which yielded higher values after water extraction compared to those after ethanol extraction. The values for worts after hops are highest with the respect to the previous worts, which indicates an increase in molecules able to influence FRAP values during the brewing process. Waste hops had particularly high values when extraction was carried out in the water (values ranged from 88 to 103 μmol TE/g）， while they were far lower when it was carried out in 70°ethanol (values ranged from 29 to 33 mol TE/g). Starting yeast exhibited the highest value （71.045±5.859 μmol TE/g）when extraction was carried out in the water， but the lowest value for 70°ethanol extracts (44.494±0.501 umol TE/g). Once again, FRAP values for waste yeasts were higher than starting values (from 103 to 136 mol TE/g for water extracts and 70 to 82 μmol TE/g for 70°ethanol extracts）， indicating that the vast was enriched with molecules able to influence FRAP analysis during the brewery process. Beers after yeast exhibited higher values than the worts in the previous stage, after boiling with and the removal of hops, indicating that when beers are in contact with yeast, they become enriched with molecules that can influence FRAPvalues. Final beers also exhibited higher values than beers in the previous stage after yeast addition, fermentation, and removal, indicating that maturation can lead to an increase in the number of molecules that can influence FRAP analysis.

In several cases, it was observed that spent yeast showed higher values than starting yeast. One possible explanation for this is that yeast may be able to absorb molecules from other materials during the brewing process and promote a release of aglycones that are more reactive than corresponding glycosides 27,28]. The fact that, in beers, an increase in FRAP values is observed with the respect to the previous wort may be due to the presence of vast that is not completely removed from the beer, which partially continues the fermentation process by releasing aglycones, which are more reactive than corresponding glycosides, as explained before. 3.4. Bioactivity of Spent Extracts in Human Keratinocytes

The bioactivity was evaluated in spent extracts, particularly in those recovered under Alter brewery. We initially evaluated the cytotoxicity of the spent malt (SP-M), spent hop (SP-H), and spent yeast (SP-YE) extracts in keratinocyte HaCaT cells. HaCaT cells were treated with extract concentrations ranging from 0.003 to 3 mg/mL for 24 h and cell viability was evaluated by MTT assay. The treatment of HaCaT cells with the extracts at concentrations lower than 0.3 mg/mL did not affect cell viability (Figure 2). The concentration of 0.03 mg/mL was, therefore, selected for the subsequent experiments. Skin aging is a complex process involving both internal and external factors, which leads to a progressive loss of cutaneous function and structure [29]. There is increasing evidence that mitochondrial dysfunction and oxidative stress are key features in skin aging [30]. In this regard, the development of ingredients that improve mitochondrial activity and prevent oxidative stress is, therefore, a potential skin anti-aging strategy.

To evaluate the ability of the extracts to improve mitochondrial activity, HaCaT cells were treated with extracts in solution without nutrients for cellular metabolism. As shown in Figure 3, the treatment of HaCaT cells for 4 h with the solution and without nutrients significantly decreased the mitochondrial activity.

Under the same experimental conditions, the addition of 0.03 mg/mL SP-H and SP-YE, but not SP-M, significantly recovered mitochondrial activity, suggesting their ability to support the mechanisms of cellular nutrition. At the same concentration, SP-M, SP-H, and SP-YE extracts were also evaluated for their antioxidant activity in HaCaT cells. HaCaT cells were treated with the extracts simultaneously or 2 h before the oxidative stress(100uM H, O, for 30 min), and the antioxidant activity was evaluated in terms of intracellular ROS formation. This experimental approach allowed for the discrimination of the ability of the extracts to counteract and/or prevent intracellular ROS formation. All the SP-M, SP-Hand SP-YE extracts directly counteracted H, O, action with a significant reduction in ROS formation in HaCaT cells (Figure 4).

4. Conclusions

In the present study, the total phenol content and antioxidant activities of different types of beers, starting materials, intermediates of the brewing process, and spent malts, hops, and yeasts were evaluated. As noted by Zhao et al. [5], the differences in the results of the analyses of antioxidant activity should be viewed in the light of differences in the analytical methods used to evaluate these activities. Differences in the results of the analyses of antioxidant activity could also be due to variations in the processes and extraction methods, and varying reaction kinetics [31]. In addition, some differences between the samples depend on their composition and not on the brewing process, given that the same process was used for all the beers. This study offers evidence that beers become enriched in phenols from their ingredients, and that brewing products and waste are interesting sources for the preparation of dietary supplements and cosmetics. This study shows the anti-aging effects of waste products from handcrafted beers in human keratinocyte cells, suggesting their potential use as ingredients for the preparation of cosmetics. Thus, this study further confirms the interest in exploiting waste from food production. Future studies will be devoted to the study and development of new, finished cosmetic formulations from beer by-products in order to investigate their possible industrial cOsmetic use.

This article is extracted from Cosmetics 2021, 8, 96. https://doi.org/10.3390/cosmetics8040096 https://www.mdpi.com/journal/cosmetics