Influence of Kiwifruit Extract Infusion on Consumer Sensory Outcomes of Striploin (M. longissimus lumborum) and Outside Flat (M. biceps femoris) from Beef Carcasses

Actinidin is a cysteine protease enzyme which occurs in kiwifruit and has been associated with improved tenderness in red meat. This study evaluated the impact of actinidin, derived from kiwifruit, on consumer sensory outcomes for striploin (M. longissimus lumborum) and outside flat (M. biceps femoris). Striploins and outside flats were collected from 87 grass-fed steers. Carcasses were graded to the Meat Standards Australia (MSA) protocols. Striploins and outside flats were then dissected in half and allocated to one of the following two treatments: (1) not infused (control) and (2) infused with a kiwifruit extract (enhanced), and then prepared as grill and roast samples. Grill and roast samples were then aged for 10 or 28 days. Consumer evaluations for tenderness, juiciness, flavor, and overall liking were conducted using untrained consumer sensory panels consisting of 2080 individual consumers, in accordance with the MSA protocols. These scores were then used to calculate an overall eating quality (MQ4) score. Consumer sensory scores for tenderness, juiciness, flavor, overall liking, and MQ4 score were analyzed using a linear mixed-effects model. Kiwifruit extract improved consumer scores for tenderness, juiciness, flavor, overall liking, and MQ4 scores for striploins and outside flat (p < 0.05). These results suggest that kiwifruit extract provides an opportunity to improve eating experiences for consumers.


Introduction
A challenge to the beef industry has been to provide consumers with a consistent and enjoyable eating experience. Lyford et al. [1] established that consumers are willing to pay higher premiums for higher quality beef. In Australia, the eating quality of beef is underpinned by the Meat Standards Australia (MSA) cut-based grading program [2]. The development of eating quality predictors was conducted through numerous consumer sensory panels evaluating the tenderness, juiciness, flavor, and overall liking of 39 cuts from beef carcasses. These cuts can be prepared using up to eight different methods [3], providing eating quality predictions for 135 cut × cook method combinations [2]. Traditionally, eating quality predictions have been used to assign one of four quality grades consisting of: (1) unsatisfactory, (2) "3 star" good everyday quality, (3) "4 star" better than everyday quality, injected at a rate of approximately 10% initial weight using a Fomaco Machine equipped with 4 mm needles (Copenhagen, Denmark). Post-enhancement muscle samples were reweighed to determine change in muscle weights associated with the enhancement process.
Enhanced and control striploins and outside flat samples were then prepared as grill (GRL) and roast (RST) samples, as per the MSA protocols as described by Watson et al. [14,15]. Briefly, GRL and RST samples were portioned into 75 × 25 × 150 mm and 75 × 75 × 150 mm portions, respectively. Grill samples were then individually wrapped in freezer film prior to packing into vacuum sealed bags [15]. Roast samples were individually packed into labelled vacuum-sealed bags [15]. Samples were then aged at 1 • C until 10 or 28 days after slaughter (4 and 22 days post-infusion treatment) at which point they were frozen and stored at −20 • C until being thawed for consumer sensory testing.

Consumer Sensory Testing
Consumer sensory testing sessions were conducted using the MSA protocols, as described by Watson et al. [14,15]. Briefly, GRL samples were cooked on a Silex grill (Silex S-Tronic Single Grill, Piotis Pty Ltd., Marrickville, Australia) heated to approximately 200 • C. Grill samples were cooked on the Silex in a predetermined order for 5 min to ensure samples were cooked to a medium doneness, samples were then rested for 2 min as per the MSA protocols, as described by Watson et al. [14,15]. Samples were then halved and served to two consumers.
Roast samples were presented to untrained consumers in three presentations (i) as a hot roast (RST) sample as per standard MSA protocol, portioned into a 10 mm slice [15]; (ii) cold roast sample portioned as a 10 mm slice (RST 10 ); and then (iii) cold roast sample portioned as a 2 mm slice (RST 2 ). Roast samples were cooked in a commercial fan-forced gas oven at 160 • C until samples reached an internal temperature of 65 • C. Samples were stored in a Bain Marie for a minimum of 5 min until preparation for serving. Samples were then trimmed to a standard size of 65 × 65 × 110 mm, before being returned to a Bain Marie maintained at 48 • C. Prior to service, the RST samples were portioned into 10 mm slices. The remaining roast samples were placed in a specifically designed roast holder and chilled overnight, and then served cold to a different consumer group (the following day). This was identical to the hot service, where RST 10 samples were prepared into 10 mm slices and offered to a second group of consumers. Similarly, at the conclusion of this service the final remaining section of the roast was prepared as a RST 2 on a deli slicer, set at 2 mm, then served to a third group of consumers, two days after the initial hot roast sensory testing. Excluding these exceptions, other procedures followed the MSA protocols as described by Watson et al. [14,15].
Grill and RST samples were presented in a controlled 6 × 6 latin square design ensuring that each sample was presented an equal number of times in the serving order from two to seven and an equal number of times before and after each of the other products, effectively balancing for potential order or halo effects [15]. Samples were prepared over 35 consumer sensory sessions consisting of 60 untrained consumers in each session, suggesting 2100 consumer scores. However, there were 20 untrained consumers that did not consume all sensory samples, thus a total of 2080 individual consumers were used within the current study. Consumers (n = 2080) evaluated each sample served for tenderness, juiciness, flavor, and overall liking by scoring a line on a 100 mm visual analogue scale ranging from 0-100. The visual analogue scale was anchored by descriptions which were not tender/very tender, not juicy/very juicy, and dislike extremely/like extremely for both liking of flavor and overall liking scores, i.e., 0 indicated a not tender sample and 100 was used to describe a very tender sample.

Statistical Analysis
The overall eating quality score (MQ4) was determined from the consumer tenderness, juiciness, flavor, and overall liking [6]. Tenderness, juiciness, flavor, and overall liking scores were weighted by 0.4, 0.1, 0.2, and 0.3, respectively, providing a MQ4 score between 0 and 100 [6]. The raw means of each sensory trait were calculated together with clipped means calculated by removing the highest and lowest 2 scores for each trait [6].
Initially, correlations between raw and clipped consumer sensory scores for meat tenderness, juiciness, flavor, overall liking, and MQ4 score were conducted. Raw and clipped consumer sensory scores for meat tenderness, juiciness, flavor, overall liking, and MQ4 score were analyzed using a linear mixed-effects model in the "lme4" package [20] and estimated marginal means were generated using the "emmeans" package [21].
Models incorporated muscle, number of days aged, treatment and cooking method, and their interactions as fixed effects. Models were refined to remove relevant insignificant interactions in a step-wise manner. The final models for tenderness, juiciness, flavor, overall liking, and MQ4 included muscle, number of days aged, treatment, cooking method, muscle × treatment, number of days aged × treatment, muscle × treatment, muscle × cooking method, number of days aged × cooking method, treatment × cooking method, and number of days aged × treatment × cooking method. Additionally, an individual animal/carcass identification was incorporated as a random effect in all models, to account for animal factors. The term cooking method was used to describe GRL, RST, RST 2 , and RST 10 .

Carcass Traits
All 87 carcasses were graded as per the MSA grading specifications [13]. Carcass characteristics as evaluated by the MSA carcass grading specifications are summarized in Table 1.

Kiwifruit Extract Infusion
The average weight increase post kiwifruit extract enhancement was 11.2% ± 0.29%, however, there was some variability in the enhancement rate between striploin and outside flat muscles ( Table 2).
Consumer sensory scores indicated that the juiciness of enhanced GRL samples that were aged for 10 days, were 19.3 points higher than control samples. Whereas juiciness scores for enhanced GRL that were aged for 28 days, increased by 7.4 points. The kiwifruit extract increased the juiciness scores of RST, RST2, and RST10 that were aged for 10 days by 11.9, 7.2, and 5.8 points, respectively, when compared with the control samples. Although the consumer evaluations of juiciness exhibited an increase of 15.9, 14.7, and 12 points in tenderness scores of enhanced RST, RST2, and RST10 that were aged for 28 days, relative to the control samples.
Flavor scores highlight that enhanced GRL samples that were aged for 10 days, were 17.6 points higher than control samples, however, 28 days aging only observed an increase of 6.3 points. Similar increases in consumer scores were observed for enhanced RST (10 days, 10 points and 28 days, 8. There was an increase of 19.5 points in MQ4 scores of enhanced GRL samples aged for 10 days, relative to the control samples. However, this did not persist to 28 days aging where the increase in MQ4 was 8.6 points. Additionally, there were increases in MQ4 scores of enhanced RST (10 days, 11.4 There were a number of days aged × treatment × cooking method influences (Figures 2 and 3) on consumer evaluations of tenderness (p = 0.005), juiciness (p < 0.001), flavor (p = 0.003), overall liking (p = 0.001), and MQ4 (p = 0.0007). However, consumer tenderness evaluations had similar increases for RST, RST 2 , and RST 10 samples aged for 10 and 28 days. At 10 days aging there was an increase of 13.6, 10.8, and 8 points for enhanced RST, RST 2 , and RST 10 as compared with the control samples. Comparably, at 28 days aging, consumer evaluations for tenderness increased 14.6, 14.2, and 12 pointsfor the enhanced RST, RST 2 and RST 10 .
Consumer sensory scores indicated that the juiciness of enhanced GRL samples that were aged for 10 days, were 19.3 points higher than control samples. Whereas juiciness scores for enhanced GRL that were aged for 28 days, increased by 7.4 points. The kiwifruit extract increased the juiciness scores of RST, RST 2 , and RST 10 that were aged for 10 days by 11.9, 7.2, and 5.8 points, respectively, when compared with the control samples. Although the consumer evaluations of juiciness exhibited an increase of 15.9, 14.7, and 12 points in tenderness scores of enhanced RST, RST 2 , and RST 10 that were aged for 28 days, relative to the control samples.
Flavor scores highlight that enhanced GRL samples that were aged for 10 days, were 17.6 points higher than control samples, however, 28 days aging only observed an increase of 6.3 points. Similar increases in consumer scores were observed for enhanced RST (10 days, 10 points and 28 days, 8.2 points), RST 2 (10 days, 10.5 points and 28 days, 10.2 points), and RST 10 (10 days, 7.5 points and 28 days, 8.7 points) samples aged for 10 and 28 days.
At 10 days aging, there was an increase of 19.6 points in consumer overall liking for enhanced GRL samples, relative to the control samples. Although, increasing the number of days aged to 28 days only increased the overall liking of enhanced GRL samples by 8.4 points. Consumer overall liking scores had similar increases for enhanced RST (10 days, 10.2 points and 28 days, 10.4 points), RST 2 (10 days, 10.2 points and 28 days, 10.7 points) and RST 10 (10 days, 7.3 points and 28 days, 9.5 points) samples aged for 10 and 28 days, when compared with the control samples.
There was an increase of 19.5 points in MQ4 scores of enhanced GRL samples aged for 10 days, relative to the control samples. However, this did not persist to 28 days aging where the increase in MQ4 was 8.6 points. Additionally, there were increases in MQ4 scores of enhanced RST (10 days, 11.4 points and 28 days, 11.5 points), RST 2 (10 days, 10.2 points and 28 days, 12 points) and RST 10 (10 days, 7.4 points and 28 days, 10.5 points) samples aged for 10 and 28 days, in comparison to the control samples.

Discussion
This study is the first to evaluate the influence of infusing beef cuts with actinidin derived from kiwifruit on untrained consumer sensory panels. The results from this study show that infusing striploins and outside flats with a kiwifruit extract improved consumer scores for tenderness, juiciness, flavor, overall liking, and MQ4 scores, which supported the initial hypothesis. Specifically, there was an increase of 13.4 points in the MQ4 score of enhanced striploins, which related to a quality grade increase from a 3-star good everyday product to a 4-star better than everyday quality product [4,5]. In comparison, outside flats had an increase of 9.3 points in the MQ4 score, which indicated a quality grade increase from unsatisfactory to a 3-star good everyday product [4,5]. Therefore, it is clearly evident that the kiwifruit enhancement was capable of improving consumer acceptance of beef striploins and outside flats, providing an opportunity for the beef industry to generate greater revenue from these primals.
Similarly, for GRL, RST, RST 2 , and RST 10 infusion with kiwifruit extract increased the MQ4 scores from 45.8 ± 0.78, 45.0 ± 1.10, 50.4 ± 1.97, and 41.9 ± 1.97 to 59.8 ± 0.85, 56.4 ± 0.85, 61.5 ± 1.36, and 50.9 ± 1.36. This equates to an increase in the eating quality predictions of RST and RST 10 from an unsatisfactory product to a 3-star good everyday grade [4,5]. However, for GRL and RST 2 , kiwifruit extract improved the eating quality from a low grade 3-star good everyday quality to a higher good everyday quality [4,5]. This suggests that the kiwifruit extract improved the overall eating experience of consumers while not impacting on the flavor of striploins or outside flats. Similarly, Christensen et al. [12] highlighted similar findings in pork M. biceps femoris. The authors reported that an improvement in textural attributes, while juiciness, flavor, and taste were not influenced by actinidin infusions [12]. Overall, this suggests that kiwifruit extract can be used to effectively tenderize meat without adversely affecting other sensory attributes.
At 10 and 28 days aging, enhanced GRL, RST, RST 2 and RST 10 exhibited increases in consumer evaluations of tenderness, juiciness, flavor, overall liking, and MQ4 scores, as compared to the control samples. However, tenderness, juiciness, flavor, overall liking, and MQ4 scores for enhanced samples aged for 28 days generally decreased in comparison to enhanced samples that were aged for 10 days, or four days post infusion. Regardless, beef infused with the kiwifruit solution and aged for 28 days still had higher consumer sensory scores as compared with the control samples. The mechanisms that resulted in the decreased consumer scores for enhanced samples aged for 28 days as compared with 10 days are yet to be clarified.
Lewis and Luh [10] indicated that actinidin may hydrolyze muscle proteins that are associated with toughness in beef cuts, rather than having a hydrolyzing action on all of the proteins in beef muscle. The tenderization of beef is a complex process that involves numerous structural changes in myofibrillar components associated with proteolytic enzymes [9]. This suggests that the improvement of eating quality from kiwifruit extracts may be due to an increased proteolytic activity on muscle proteins [11], possibly specifically myofibrillar proteins [8], by actinidin.
The results presented here suggest that proteolysis is sustained in beef cuts infused with kiwifruit extracts up to 28 days and possibly longer. This suggests that there may be a risk of over tenderization occurring due to the continued proteolytic activity [22]. Hence, aging beyond 10, or 14 days [8], may not provide the optimum eating experience for consumers. As such, these results suggest that the optimum number of days for aging beef infused with kiwifruit extracts warrants further investigations. Furthermore, it is important to consider that the optimum number of days for aging may vary across different cuts of meat, as some beef cuts may benefit from a longer period of aging [14,23]. Understanding the optimum number of days for aging post infusion process would ensure that a consistent and enjoyable eating experience for consumers is achieved.
This study has highlighted that using a kiwifruit infusion improved the quality grades of striploin and outside flats within this study, suggesting that kiwifruit extract provides an opportunity to improve the value of low-and poor-quality beef. Lyford et al. [1] indicated that consumer willingness to pay increased by approximately 50% for increased quality of (1) unsatisfactory to 3-star good everyday quality and (2) 3-star good to 4-star better everyday quality beef. Therefore, developing value adding methods for improving the quality grades of low-quality cuts is important for the long-term financial sustainability of the beef industry. On the basis of these findings, it could be reasoned that the kiwifruit infusion of low-quality striploins and outside flats has the potential to "double" the market value within Australia. Nevertheless, these results suggest that cuts infused with the kiwifruit solution should be cooked or frozen within a short period after the infusion process is conducted to reduce the activity of actinidin and ensure an optimum consumer experience.

Conclusions
Enhancing striploins and outside flats improved the eating quality scores of GRL, RST, RST 2 , and RST 10 as evaluated by untrained consumers. In both muscles, at 10 and 28 days post-mortem aging, consumed as GRL or RST, the tenderness, juiciness, flavor, and overall liking of the kiwifruit enhanced product was increased, improving the intrinsic eating quality. As such, this study suggests that kiwifruit extract has the potential to increase consumer acceptance while increasing profitability of processors by improving unsatisfactory and low-quality beef cuts into acceptable and high-quality cuts.
Author Contributions: R.P., M.K., and P.M. contributed to the conceptualization and experimental design of the study; M.K., P.M., and R.P., undertook the study; A.L. and G.T. conducted the formal data analysis and presentation of the data; A.L. wrote the original manuscript draft; reviews and editing of the completed manuscript was conducted by G.T., M.K., R.P., and P.M.; project administration and funding acquisition was the responsibility of R.P. and P.M.
Funding: This research was funded by the Meat and Livestock Australia P/L., North Sydney, NSW, Australia; grant number: NBP.0722, "Transport Duration Effects on MSA Eating Quality".