Effect of alternative pre-treatments and fermentation on quality characteristics of oyster mushrooms

Besides their unique taste and texture, mushrooms are a promising source of important nutrients, including dietary fiber, amino acids, minerals, and vitamins. Fresh mushrooms, however, can only endure for a brief time, typically up to three days at ambient conditions. Different methods have been used to preserve mushrooms for a prolonged period, such as drying, cooking, frying, irradiation and fermentation. The objective of the current study is to investigate the effect of different pre-treatments and fermentation on physicochemical, textural, and microbial properties of oyster mushrooms. The fresh oyster mushroom was considered as control and 6 alternative pre-treatment methods were used as; blanching in water, steaming, oven cooking, microwave, High Hydrostatic Pressure and UV Light treatment. Moisture, pH, yield, color, texture, and microbiological analyses were performed on each pre-treatment group before and after fermentation. Our results showed that the quality attributes of oyster mushrooms were significantly affected by the usage of different pre-treatments.


INTRODUCTION
Edible mushrooms are a very diversified group of fast-growing macrofungi and popular food items worldwide.Mushrooms have been consumed and studied for their several nutritional, medicinal, economical, and sustainable contributions and are considered next-generation healthy foods (Bringye et al., 2021;Das et al., 2021;Kumar et al., 2022).They can play a significant role in adaptive immunity, therapeutic attributes, and improvement of general health thanks to the primary components of mushroom polysaccharides, such as non-digestible prebiotic β-glucans (Kiss et al., 2021).Pleurotus mushrooms, also known as "oyster mushrooms" are one of the members of Basidiomycota and they are recognized for their nutritional and culinary value.They can grow on various agricultural wastes in a short growth time and can be cultivated easily and affordably as they do not require specific environmental control (El-Ramady et al., 2022).Pleurotus ostreatus, thanks to high protein and essential amino acid content, could be regarded as a viable vegan-protein source (Bakratsas et al., 2023).Besides being low in fat, calories, and cholesterol, they are rich in bioactive compounds such as β-glucans, phenolic compounds, secondary metabolites, dietary fiber, vitamins, and carbohydrates (Sharma et al., 2021).On the other hand, fresh mushrooms are prone to quick decay primarily because of the high water content (87-95%), metabolic rate, enzyme activity, and susceptibility to bacterial attack.They start deteriorating immediately after harvest and have a limited shelf life at ambient conditions, which might result in a loss of quality (moisture loss, discoloration, alteration of texture and flavor and reduction in nutrient content) and thus rejection by consumers (Nketia et al., 2020).Various preservation techniques such as drying, cooking, pickling, frying, and gamma irradiation have been investigated and utilized to extend mushroom shelf life and diversify the product for consumers (Maray et al., 2017;Nketia et al., 2020).Preservation of mushrooms by fermentation is also an affordable and efficient method domestically used in Eastern Europe and Asia (Jabło nska-Ry s et al., 2016).The current study aims to investigate the effects of different pre-treatments and fermentation on physicochemical, textural and microbiological properties of oyster mushrooms.The applied processing used in this study included blanching in water, steaming, oven cooking, microwave, High Hydrostatic Pressure and Ultraviolet Light treatment, and spontaneous fermentation of oyster mushrooms.

Pretreatments and fermentation
The fresh oyster mushroom (P.ostreatus) utilised in this research was supplied from a local market in Budapest, Hungary.The pre-treatments were performed at the pilot plant of the Hungarian University of Agriculture and Life Sciences (MATE), Department of Livestock Products and Food Preservation Technology.The damaged parts of oyster mushroom were separated.Cleaned, and longitudinally sliced mushrooms were used for pretreatments and fermentation.2 kg of fresh oyster mushroom was used for each pretreatment group.Six different pretreatments applied to oyster mushrooms before fermentation were as follows; blanching in water (B), steaming (S), oven cooking (O), microwave (M), High Hydrostatic Pressure (H) and Ultraviolet Light treatment (U) and fresh oyster mushroom was used as control (F) (Table 1).
Images of the pretreated mushroom samples are presented in Fig. 1.After the pretreatments, the fermentation process was applied in accordance with a prior investigation by Jabło nska-Ry s et al. ( 2022), with some modifications.All pretreated sample groups were divided into portions of 250 g each and left to spontaneous fermentation for 8 days at 21-22 ºC in sealed pouches with the addition of 2% (w/w) of salt, 1% (w/w) of sucrose and 70 ml of 2% salt solution.Images of the fermented mushroom samples are presented in Fig. 2. Fresh mushrooms, pretreated mushrooms, and mushrooms that were fermented for 8 days were evaluated in terms of moisture, pH, color, texture, Lactic acid bacteria count and yield were calculated for each group.All treatments were repeated twice independently.Moisture content, pH, yield and LAB count The moisture content of mushroom samples was assessed in duplicate in accordance with AOAC, 2000.For this purpose, approximately 4 g of mushroom was dried in a convection oven (Labor MM, Hungary) at a temperature of 105 8C throughout 16 h.The pH of the mushroom samples was assessed with a pH meter (Testo AG., Germany) in triplicate.The weight of the mushrooms before and after the pretreatments and fermentation were recorded, and afterwards, the yield for each respective group was calculated according to the given formula.
Yield ¼ Initial weight of the sample = Final weight of the sample X 100 The following method was used to enumerate the Lactic Acid Bacteria (LAB) count: United States Pharmacopeia (USP) (2013).

Color measurement
The color characteristics of mushroom samples were measured employing the CIELAB (Colorimetry, C. I. E., 1986) scoring system.Lightness (L p ), redness (a p ), and yellowness (b p ) values of the mushroom samples were determined using a CR-410-type colorimeter (Konika Minolta Sensing Inc., Japan).Prior to each measurement, the colorimeter was calibrated using a white standard plate (CRA43).Nine parallel readings were performed for each mushroom sample.Browning Index, Yellowness Index, and Total Color Change (ΔE) were calculated according to Doro ski et al. ( 2020).

Texture properties
The texture of mushroom samples was evaluated witha TA.XTplus Texture Analyzer (Stable Micro System, Surrey, UK). 60 g of mushroom samples were positioned within a Kramer cell for the shear force analysis.The speed for both pre-measurement and during measurement was fixed as 2 mm s À1 , while the distance was 30 mm.Trigger force was set as 0.049 N. Force (N) was recorded as a function of time/distance.The obtained peak force (N) to shear through the sample was used to determine the firmness of the sample.Measure of work (mJ) performed during each trial was calculated assessing the obtained graph.Nine shear press values were derived for each pretreatment group.

Statistical analysis
One-way multivariate ANOVA in SPSS-23 software (SPSS Inc., IBM Company, US) was used for the statistical evaluation of the obtained results.Levene's test was used to confirm the homogeneity of the measured values.Tukey's post hoc tests were conducted for identifying any significant differences among the samples.The differences were regarded to be statistically significant at P < 0.05.

Moisture content
The moisture contents of the mushroom samples after pretreatments and after fermentation are displayed in Table 2. Use of different pretreatments caused significant differences in the moisture content of the mushrooms.After pretreatments, B, H, O, and U samples had similar moisture contents, ranging between 88.90 and 90.47%, being close to the moisture content of fresh oyster mushrooms (89.66%) (Nketia et al., 2020).The lowest moisture content was obtained as 83.91% for M samples while S samples had 87.16% moisture content.Significant differences in the moisture content of the mushroom samples were also observed after the fermentation.F and U samples after fermentation had similar and highest moisture content (90%), and M samples had the lowest (85.56%).Similar results were reported by Wang (2015) in their research of Auricularia auricula mushroom pickles.The highest moisture content in U samples could be explained by the fact that UV treatment a surface treatment.The lowest moisture content in M samples might be attributed to water molecules in mushrooms absorbing microwave energy quickly, whereas microwave energy was converted to heat, causing water to evaporate rapidly (Zhang et al., 2018).

pH
The pH value of fermented foods is a critical element in ensuring product durability and microbiological safety.As a result, to achieve a rapid and significant decrease in pH (increase in titratable acidity) in fermented foods is critical.The fresh oyster mushroom that was used in our study had a mean pH of 6.35 ± 0.04.The pH values of pretreated and fermented mushroom samples are given in Table 2. Different pretreatments and fermentation had a significant impact on the pH of the mushroom samples.The pH values of control sample (F) were the lowest while the B samples had the highest pH value, 6.35 ± 0.04 and 7.60 ± 0.02 respectively.All pretreated samples had a higher pH value compared to fresh oyster mushrooms.Similarly, LeLAS et al. (2007) reported an increase in the pH of button mushrooms after water blanching for 3 min.As expected, all sample groups had a pH drop after fermentation.The lowest pH after fermentation was achieved in F samples as 3.96 ± 0.07 and the highest pH was detected in S samples as 4.98 ± 0.25.These results are consistent with the findings of Chen et al. (2021) where the pH of Shiitake mushroom was dropped from 6.5 to 5.0 after 5 days of fermentation.In another study with oyster and chanterelle mushrooms, after 3 or 4 days of fermentation, with L. plantarum, pH values reached 3.5 (Jabło nska-Ry s et al., 2016).According to research, the pH of other fermented mushroom species ranges between 3.3 and 4.6 and it is regulated by fermentation temperature, carbohydrate content, and chemicals employed in the process (Jabło nska-Ry s et al., 2019).

Yield
The yields of applied pretreatments before and after fermentation are given in Table 2.For fresh mushrooms (F), it was estimated as 100% as no pretreatment was applied to these samples as a control group.The final weight of mushroom after pretreatments was found highest for the U (99.71%) and H samples (98.65%).Among the pretreatments, mushrooms obtained after microwave treatment revealed the lowest yield (80%).The loss in mushroom weight of M, S, and O samples may be due to the removal of water, while it could be to the solid waste from the mushroom tissues for B samples (Maray et al., 2017).After the fermentation, M samples exhibited the highest yield (95.46%), which was followed by B (92.32%) and S (89.59%) samples.The lowest yield was obtained for H (68.70%) and U (69.41%) samples after the fermentation.This result points out to the importance of pretreatments before fermentation process in terms of yield.

Lactic acid bacteria count
Lactic acid bacteria counts of the mushroom samples after pretreatments and fermentation are shown in Table 3.The highest number of LAB was recorded for U and F samples after pretreatments, i.e., 2.54 and 2.29 log CFU mL À1 respectively.The population of LAB in all pretreated fermented mushroom samples increased by the end of fermentation.A favorable population of LAB (>8 Log CFU mL À1 ) was acquired for four pretreatment groups (S, H, B, O) after 8 days of fermentation.Similar results were reported by Jabło nska et al. ( 2022) with fermented button mushrooms.Low pH, high organic acid concentrations, and a decrease in the accessible carbohydrates amount all have an inhibitory effect on the LAB population.

Color
Mushrooms are prone to browning and color change due to processing, microbial contamination and enzymatic activity.Pretreatments and fermentation significantly affected the color of the mushrooms (Table 4).Compared to untreated samples (F), all pretreated samples had significantly lower L p values, other than U samples (P < 0.05).This was in accordance with the Browning Index, indicating that the browning occurred the most in S, M and O, and the least in U samples.Similar results were reported by Wang et al. (2017) for UV-C treatment, averting surface browning of oyster mushrooms.Yellowness Index followed the same pattern with the Browning Index, in accordance with b p values.The lowest a p values were detected for H and O samples.Total color difference (TCD), evaluating the complete variations in color of an analyzed sample in comparison with a control (in our study, F), indicates that the H and S samples experienced the biggest color changes.Similarly, darkening and significant changes in BI were observed for canned oyster mushrooms after steam blanching.It was elucidated as the outcome of the enzymatic oxidation of polyphenols by polyphenol oxidase in mushrooms, causing browning and subsequent loss of whiteness (Nketia et al., 2020).Our results are in agreement with Eissa et al. (2009) where the water-blanched mushrooms had a BI lower than steam-blanched mushrooms, due to thermal pretreatments increasing the development of nonenzymatic browning as in the case of steamed, microwaved and oven treated mushrooms in our study.
After the fermentation process, significant differences were detected among the samples regarding all color attributes.The L p values detected as the highest for B and M samples and lowest for F, U, O and H samples.This was in accordance with BI indicating that blanching in water and microwave treatments could prevent the browning of mushrooms.Similarly, B samples showed the highest a p values compared to other samples, followed by S and H samples. F, M, U and O samples experienced the biggest losses of redness.YI in agreement with b p values showed that H samples exhibited the highest yellowness while B samples had the lowest.It was also observed that fermentation caused a decrease in lightness and redness values (other than B samples) with a high increase in yellowness values of all mushrooms.Liu et al. (2016) reported similar results for Pleurotus spp.subjected to lactic fermentation where they reported a comparable rise in b p values, in addition to darkening of the fruiting bodies (decreased L p value), however an increase in the a p values was also reported in their study.In another study with button mushrooms, an increase in L p and b p values was observed with a decrease in the values (Jabło nska-Ry s et al., 2022).

Texture
The texture of mushrooms is subject to change due to time, loss of water, wounds, mechanical damage, and thermal processing (Zhang et al., 2018).Also, the application of heat and the Progress in Agricultural Engineering Sciences 19 (2023) S1, 35-45 specific methods employed for thermal processing are of important factors (Hasani et al., 2021).The type of pretreatment applied before fermentation led to significant differences in textural parameters of mushrooms (P < 0.05).The results of texture evaluation are demonstrated in Fig. 3a and b.According to results, the force required to shear F (54.79 ± 4.86), U (54.41 ± 3.10) and M (52.41 ± 3.39) samples was much higher than that of other samples, while H (35.30 ± 4.39) samples required the lowest.Also, more shear work was required to cut through these samples than H and B samples Fig. 3(a).In a study with Agaricus bisporus and Boletus edulis mushrooms, Kramer shear cell measurement indicated that the shear force for blanched Agaricus mushrooms was higher than that of fresh ones, while the shear force for blanched Boletus mushroom was lower compared to the fresh ones.Work values were also detected to be higher for fresh mushrooms compared to blanched mushrooms (Jaworska et al., 2010).Similar results were explained by high temperatures while blanching, causing denaturation of proteins, membrane destabilization, and decrease in weight or volume leading to softening of mushroom tissues (Zivanovic et al., 2004).It is noteworthy that in our study, H samples experienced more textural alterations even compared to heat treated samples while U and M samples demonstrated much better results.This outcome could potentially be attributed to the fundamental distinctions in how heat treatments and HHP treatment impact proteins (Kenesei et al., 2017).There were significant differences between the samples after fermentation (Fig. 1(b)).The force required to shear B (46.85 ± 4.29), M (46.37 ± 4.85) and S (44.88 ± 5.35) mushrooms after the fermentation process was higher than that of other samples, while U (8.53 ± 1.12) samples required the lowest force.Shear work values were in accordance with the required force after the fermentation as well, less work was required to cut through U, H, and F samples after fermentation.In their study, Jabło nska-Ry s et al. (2022) reported that fermentation of the mushroom fruiting bodies resulted in reduced firmness, pointing out that it is a common problem for fermented vegetables.In our study, the objective was to see the effects of different pretreatments on mushrooms before and after fermentation.As a result, blanched, steamed and microwave-treated mushroom samples were able to tolerate both pretreatment and fermentation processes with less change in texture.The biggest alterations were observed in fresh, UV, and HHP-treated mushrooms.

Fig. 3 .
Fig. 3. (a): Texture evaluation of the mushroom samples after pretreatments; (b): Texture evaluation of the mushroom samples after fermentation.a-d, AÀE : Mean values with different letters indicate significant differences among samples (P < 0.05)

Table 1 .
Pretreatment of oyster mushroom

Table 2 .
Moisture, pH and yield values of the mushroom samples after pretreatments and after fermentation

Table 3 .
Lactic Acid Bacteria (LAB) counts of mushroom samples

Table 4 .
Means ± standard deviations of color attributes of mushroom samples, Browning Index, Yellowness Index and Total Color Change (ΔE) a-d, AÀC : Mean values with different letters differ significantly among samples (P < 0.05).